crystallographic analysis of the sec-dependent...
TRANSCRIPT
CRYSTALLOGRAPHIC ANALYSIS OF THE SEC-DEPENDENT SECRETION CHAPERONE
CsaA
Yuliya Shapova B.Sc., Simon Fraser University, 2005
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
In the Department of Molecular Biology and Biochemistry
O Yuliya Shapova 2007
SIMON FRASER UNIVERSITY
2007
All rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other means, without permission of the author.
APPROVAL
Name:
Degree:
Title of Thesis:
Yuliya Shapova
Master of Science
Crystallographic Analysis of the Sec-dependent secretion chaperone CsaA.
Examining Committee:
Chair: Dr. David Vocadlo Assistant Professor, Department of Chemistry
Dr. Mark Paetzel Senior Supervisor Assistant Professor, Department of Molecular Biology and Biochemistry
Dr. Nancy Hawkins Supervisor Assistant Professor, Department of Molecular Biology and Biochemistry
Dr. Peter J. Unrau Supervisor Associate Professor, Department of Molecular Biology and Biochemistry
Dr. Jack Chen Internal Examiner Associate Professor, Department of Molecular Biology and Biochemistry
Date DefendedlApproved: u
SIMON FRASER ' UNIVERSITY~ brary
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Simon Fraser University Library Burnaby, BC, Canada
Revised: Spring 2007
ABSTRACT
The eubacterial protein CsaA has been proposed to act as a protein
secretion chaperone in the Sec-dependent translocation pathway. Two structures
of CsaA from Bacillus subtilis were solved by X-ray crystallography and refined to
1.9 and 2.0 A resolution. Structural analysis revealed two potential substrate
binding pockets on the surface of CsaA. These pockets display biochemical
properties consistent with the substrate binding preference of CsaA. A structure
of CsaA from Agrobacterium tumefaciens in complex with a phage display
derived peptide was solved to 1.65 A resolution. The peptide binds to the
substrate binding pocket on the surface of CsaA. Three residues of the bound
peptide form specific interactions with CsaA: glutamine at position (-6) and small
hydrophobic residues at positions (-5) and (-3). A conserved arginine residue in
the binding site of CsaA likely acts as a clamp that transiently interacts with and
stabilizes the peptides in the binding site.
Keywords: chaperone, X-ray crystallography, Secdependent protein secretion, peptide binding, protein structure
iii
First of all, I would like to thank my senior supervisor, Dr. Mark Paetzel, for
giving me a wonderful opportunity to learn about the exciting field of X-ray
crystallography and protein structure, and for his continuous support and
guidance along the way. I was greatly motivated by his interest and enthusiasm
for this project. I would also like to thank my supervisory committee members, Dr.
Nancy Hawkins and Dr. Peter Unrau, for their excellent advice and suggestions.
I would like to thank Dr. Anat Feldman for teaching me the techniques of
molecular biology and biochemistry, and Dr. David Oliver and Dr. Jaeyong Lee
for patiently answering my questions.
I would like to thank our lab manager, Deidre de Jong-Wong, for making
the Paetzel lab such an organized workplace, and for her support and
encouragement. Thank you also to all the past and present graduate students in
the Paetzel lab: Chuanyun Luo, Apollos Kim, Ivy Chung, Kelly Kim, Alison Li,
Charles Stevens, and Sung-Eun Nam, for creating such an enjoyable working
environment.
Finally, I would like to thank my husband, Oleg Titov, for always believing
in me, and for his unwavering support throughout my studies.
TABLE OF CONTENTS
Approval .............................................................................................................. ii ... Abstract .............................................................................................................. 111
Acknowledgements ........................................................................................... iv
Table of Contents ............................................................................................... v ... List o f Figures .................................................................................................. VIII
List of Tables ...................................................................................................... x
Glossary ............................................................................................................. xi
Chapter 1 . An Overview of the Molecular Chaperones from a ........................................................................................ Structural Perspective 1
Introduction ......................................................................................... 1 ..................................................................................... Trigger Factor 3
SurA and MPN555 ............................................................................. 6 ............................................................................... Hsp70 and Hsp40 7
Hsp9O ............................................................................................... 12 ............................................................................... Prefoldin (GimC) 14
Skp ................................................................................................... 17 LolA .................................................................................................. 18 PapD. FimC ...................................................................................... 20 Type Ill secretion chaperones .......................................................... 25 Signal Recognition Particle ............................................................... 28 SecB and CsaA ................................................................................ 33 TorD ................................................................................................. 36 GroEL and GroES ............................................................................ 38 Group II Chaperonins ....................................................................... 42 The ClplHspl00 family ..................................................................... 45 Conclusion ...................................................................................... 48
........... Chapter 2 . The Crystallographic Analysis of Bacillus subtilis CsaA 51 2.1. Introduction ....................................................................................... 51 2.2. Materials and Methods ..................................................................... 55
.......................................................................... 2.2.1. PCR and Cloning 55 ................................................... 2.2.2. Overexpression and Purification 58
2.2.3. Crystallization and Data Collection ............................................... 60 2.2.4. Structure Determination and Refinement ...................................... 61 2.2.5. Structural Analysis ........................................................................ 62
2.3. Results and Discussion .................................................................... 63
.......................................................................... 2.3.1. PCR and Cloning 63 ................................. 2.3.2. Overexpression and Purification of BsCsaA 66
2.3.3. Crystallization and Data Collection ............................................... 70 ...................................... 2.3.4. Structure Determination and Refinement 71
2.3.5. Sequence Alignment Analysis ....................................................... 74 ....................................................................... 2.3.6. Structural Overview 75
............................................................ 2.3.7. The Dimerization Interface 78 ............................................ 2.3.8. The Potential Substrate Binding Site 81
.............................. 2.3.9. The Electrostatics and Conservation Analysis 86 2.4. Conclusion ........................................................................................ 88
Chapter 3 . Cloning, Overexpression, Purification, Crystallization, and Refinement of the Crystal Structures of Agrobacterium tumefaciens CsaA .................................................................................................................. 89
3.1. Introduction ....................................................................................... 89 3.2. Materials and Methods ..................................................................... 91
.............................. 3.2.1. PCR and Cloning of AtCsaA and X15-AtCsaA 91 ....... 3.2.2. Overexpression and Purification of AtCsaA and XIS-AtCsaA 93
3.2.3. Crystallization and Data Collection of AtCsaA and X I 5- .......................................................................................... AtCsaA 94
3.2.4. Structure Determination and Refinement of AtCsaA and .................................................................................. X I 5-AtCsaA 95
........................................................................ 3.2.5. Structural Analysis 96 .................................................................... 3.3. Results and Discussion 97
......................................................... 3.3.1. PCR and Cloning of AtCsaA 97 .................................. 3.3.2. Overexpression and Purification of AtCsaA 99 ................................. 3.3.3. Crystallization of AtCsaA and X I 5-AtCsaA 101
3.3.4. Structure Determination and Refinement of AtCsaA and ................................................................................ X I 5-AtCsaA 103
3.3.5. An Overview of the AtCsaA structure and comparison to ....................................................................................... BsCsaA 105
......... 3.3.6. Interaction of X I 5-AtCsaA with the co-crystallized peptide 109 3.3.7. A comparison of the substrate binding pockets in the
structures of CsaA from A.tumefaciens, B.subtilis, and ............................................................................ T . fhermophilus 113
...................................................................................... 3.4. Conclusion 116
Appendix A . Cloning, Purification, and Crystallization of ....................................................................................... A . tumefaciens SecB 118 ..................................................................................... A.1. Introduction 118
................................................................... A.2. Materials and Methods 118 ........................................................................ A.2.1. PCR and Cloning 118
..................................... A.2.2. Protein Overexpression and Purification 119 ............................................. A.2.3. Crystallization and Data Collection 120
A.3. Results and Discussion ................................................................ 122 ........................................................................ A.3.1. PCR and Cloning 122
A.3.2. Overexpression and Purification of AtSecB protein .................... 124
A.3.3. Crystallization and Data Collection ............................................. 126
Appendix B ...................................................................................................... 128
Reference List ................................................................................................. 184
vii
LIST OF FIGURES
Figure 1 . 1 The structures of the Trigger Factor. SurA. and MPN555 ............... 5
Figure 1.2 The structures of Hsp70 and Hsp40 ......................................... 10
Figure 1.3 The structures of Hsp9O ................................................................ 13
Figure 1.4 The structures of prefoldin and Skp ......................................... 15
Figure 1.5 The structures of LolA and LolB .................................................... 20
Figure 1.6 The structures of the type I pili chaperone FimC and the P pili chaperone PapD ............................................................................ 23
Figure 1.7 The structures of the Type Ill secretion system chaperones in complex with their effector substrates .............................................. 26
Figure 1.8 The structures of the Signal Recognition Particle ......................... 30
Figure 1.9 The structures of SecB and CsaA ................................................. 35
Figure 1 . 10 The structure of TorD ................................................................. 37
Figure 1.1 1 The structures of GroES and GroEL .......................................... 40
Figure 1.12 The structure of the archaeal thermosome from Thermoplasma acidophilum ................................................................. 44
Figure 1 . 13 The structures of the Clp/Hsp100 family chaperones ClpA and ClpB .............................................................................................. 46
Figure 2.1 A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria ............................................................... 52
Figure 2.2 The optimization of the PCR amplification of B.subtilis csaA gene ..................................................................................................... 63
Figure 2.3 The results of cloning the PCR-amplified B.subtilis csaA gene into pCR2.1 -TOP0 vector ........................................................... 64
Figure 2.4 The results of subcloning of the csaA gene fragment into the expression vector pET28.a(+) ............................................................. 65
Figure 2.6 Purification of BsCsaA protein by nickel affinity chromatography ................................................................................... 67
Figure 2.7 Optimization of thrombin digest of BsCsaA ................................... 68
........... Figure 2.8 Purification of BsCsaA by size exclusion chromatography 69
viii
Figure 2.9 Crystals of B.subtilis CsaA ............................................................ 70
Figure 2.1 1 The structure of BsCsaA ............................................................ 76
Figure 2.12 Dimerization of BsCsaA via hydrogen bonding .......................................................................................... interactions 79
Figure 2.1 3 The potential substrate binding sites in BsCsaA ........................ 82
Figure 2.14 Docking of BsCsaA structure with a peptide in extended conformation ........................................................................................ 84
Figure 2.15 The conservation and surface electrostatic properties of BsCsaA ................................................................................................ 87
Figure 3.1 PCR amplification of A.tumefaciens CsaA gene ........................... 97
Figure 3.2 Cloning of A.tumefaciens CsaA .................................................... 98
.............. Figure 3.3 Purification of AtCsaA by nickel affinity chromatography 99
Figure 3.4 Optimization of the thrombin digest reaction of A.tumefaciens CsaA .......................................................................... 100
Figure 3.5 Initial crystals of AtCsaA ............................................................. 101
.............................................................. Figure 3.6 The structure of AtCsaA 107
Figure 3.7 The structure of AtCsaA in complex with a phage-display derived peptide (XI 5.AtCsaA) ........................................................... 110
Figure 3.8 The substrate binding pockets from the structures of AtCsaA. X I 5.AtCsaA. BsCsaA. and TtCsaA ..................................... 114
Figure A1 PCR and cloning of Ahmefaciens secB gene ............................ 122
Figure A2 Overexpression and purification of A.tumefaciens SecB ............ 124
Figure B1 A phylogenetic tree based on the sequences of 18 CsaA and 18 TRBP and MetRS (C-terminal part only) ....................................... 129
Figure B2 A sample diffraction pattern and a Ramachandran plot of the ............ crystallographic model of BsCsaA in the space group P3221 130
Figure 83 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P42,2 ............ 131
Figure 84 Ramachandran plots of the crystallographic models of AtCsaA ligand-free (A) and with the symmetry related in the putative binding site (B) .................................................................... 132
LIST OF TABLES
Table 2.1 The crystallographic data collection statistics for B.subtilis CsaA. . . . .. . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Table 2.2 A summary of refinement statistics for the models of B.subtilis CsaA structure. .............................. . . . . . . . . . . ........... ......................... 73
Table 2.3 The structural neighbors of B.subtilis CsaA .................................... 78
Table 2.4 The inter-chain hydrogen bonds between the two monomers of BsCsaA ...... . . .. . .. . . ... . .. . . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Table 3.1 The data collection statistics for the structures of AtCsaA and X I 5-AtCsaA. .. .. .. . . . . ... . . .. . . .. . .. . . ... . .. .. . ... . . . . . .. . . . . . .. .. . . . . . . .. .. .. . . .. . . . . . .... . . . . . . . l o3
Table 3.2 The progress of refinement of AtCsaA and X I 5-AtCsaA structures ..... .. . . .. . . .. . .. . . .. . . .. . .. . ... . . .. ... . .. . .. . . .. . ... . . . . . . .. . .. . . .. .. .. . . . .. . .. . . .. . . .. . . lo4
Table 3.3 The refinement statistics for the structures of AtCsaA and X I 5-AtCsaA. .. . . .. ... . . .. . . .. . . . . . ... . .. .. .... . . .. . .. . . . . . .. .. . . . . . ... . . . . . . .. . . . . . ... . . . . . . . .. . . . lo5
Table 3.4 The interchain hydrogen bonds between the two monomers of AtCsaA. ...... . ... .. . . .. . . .. . . . . ... . . .. . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I08
Table 3.5 Direct hydrogen bonds between the peptide and X I 5-AtCsaA. .... 11 1
Table 61 Structures of the chaperone proteins listed in the Protein Data Bank. . . .. .. . . .. . .. . ... . .. . . .. . . .. . ... . .. .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
GLOSSARY
A Angstroms, a unit of measurement. 1A = lo-'' meters
ADP Adenosine diphosphate
Asymmetric unit The largest assembly of molecules that has no symmetry in itself, but can be superimposed on other identical elements in the unit cell by symmetry operations
ATP
B-factor
Adenosine triphosphate
A measurement of the displacement of an atom from its position due to thermal motion and conformational disorder. B-factor is elated to the displacement u by the equation:
= 8n2(u2\ High B-factors indicate a high degree of
disorder and a low degree of confidence about that particular part of the model.
Completeness The number of crystallographic reflections measured in a data set, expressed as a percentage of the total number of reflections present at the specified resolution.
Crystal An array of atoms, molecules, or ions, which are packed in a regularly ordered, repeating pattern extending in all three spatial dimensions.
Data
Dalton, a unit used to express molecular masses. 1 Da = 1 gram per mole
The positions and intensities of reflections from a single crystal in the diffraction pattern produced by X-ray crystallography
Electron Density An image of electron clouds surrounding the molecule Map
ER Endoplasmic reticulum
Hanging drop vapour diffusion
HEPES
l PTG
Macromolecular crowding
Mother liquor
Occupancy
PCR
PDB
PEG
Phage Display
A common method of protein crystallization, in which small volumes of precipitant and protein are mixed together and the drop is equilibrated against a larger reservoir of solution containing precipitant or another dehydrating agent. Drops are placed on a coverslip that seals the reservoir, such that they hang over the reservoir solution. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir until equilibration concentration is reached. This process may produce favourable conditions for crystallization.
4-(2-hydroxyethy1)-I-piperazineethanesulfonic acid
A significant volume of the cell cytosol is occupied by molecules (proteins, RNA, sugars and others). This crowding can drastically alter the kinetics or biophysical properties of molecules.
The part of a solution that is left over after the crystals are removed.
A measure of the fraction of molecules in the crystal in which a particular atom actually occupies the position specified in the model. If all molecules in the crystal are precisely identical, then occupancies for all atoms are 1.00.
Polymerase chain reaction
Protein Data Bank, http://www.rcsb.org
Polyethylene glycol
A technique used to select the peptide binding partners for the target protein. A library of variants of a peptide or protein is expressed on the outside of a phage virion. The selection process is carried out by incubating a library of phage-displayed peptides with a plate coated with the target, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional bindinglamplification cycles to enrich the pool in favour of binding sequences. After 3-4 rounds, individual clones are characterized by DNA sequencing and ELISA.
xii
Ramachandran A plot showing the main-chain conformational angles in a plot polypeptide. The conformational angles plotted are phi, the
torsional angle of the N-CA bond; and psi, the torsional angle of the CA-C bond. Due to steric repulsion, only certain conformational angles are allowed. The plot is used as a tool to assess the validity of the model.
Redundancy
Refinement
R factor
The data sets contain several independent measurements of each reflection due to symmetry in the crystal and overlap in measurements. Redundancy gives the average number of independent measurements of each reflection in a crystallographic data set and is calculated as: (number of measured reflections) 1 (number of unique reflections).
The process of improving the agreement between the molecular model and the crystallographic data by adjustment of positions, occupancies, and B-factors of atoms in the model. Progress in refinement is signified by decreasing R values, disappearance of residues from the unfavourable region of the Ramachandran plot, and improving chemical plausibility of the structure (e.g. bond lengths and angles).
A measure of agreement between the crystallographic model and the original X-ray diffraction data, calculated as
R = C I l ~ ~ ~ ~ 1 - IFc&II/CIFobsI where ~~b~ and F ~ ~ I ~ are intensities observed from the measured data or calculated from the model, respectively.
Calculated using the same equation as the R-factor, except only for a small subset (5-1 0%) of randomly chosen intensities, which are set aside from the beginning and not used in refinement. Rkee measures how well the current model predicts a subset of the measured reflection intensities that were not included in the refinement. Rfree is higher than the R factor at the beginning of refinement, but in the final stages, the two values should become more similar.
A measure of agreement among multiple measurements of the same reflections, with the different measurements being in different frames of data or different data sets. R,,,,, is calculated as follows: Rm,, = C 11 - (I)I/Z ( I ) (I is the
individual measurement of each reflection, and <I> is the
xiii
average intensity from multiple observations):
r.m.s.d. Root mean square deviation
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Sitting drop Same principles as described for hanging drop vapour vapour diffusion diffusion, except drops are placed on a pedestal above the
reservoir solution
Space group Designation of the symmetry of the unit cell of a crystal.
TRlS 2-amino-2-hydroxymethyl-1,3-propanediol
Unit cell The simplest repeating unit in the crystal that is representative of the entire crystal
xiv
CHAPTER 1. AN OVERVIEW OF THE MOLECULAR CHAPERONES FROM A STRUCTURAL PERSPECTIVE
1 .I. Introduction
Originally, the term "molecular chaperone" was used to describe a class of
proteins that assist the correct folding and assembly of other polypeptides but are
not part of the final structure (Ellis, 1987). Even though is has been demonstrated
that most proteins can assume their correct fold in vifro without the assistance of
any other factors (Anfinsen, 1973), the situation in the cell is quite different
because of the presence of numerous other proteins that produce the effect of
macromolecular crowding (Ellis & Minton, 2006). In these conditions, it may be
problematic for proteins to reach their correct fold. Therefore, many proteins rely
on molecular chaperones to create a proper microenvironment that produces
favourable conditions for their folding and prevents improper interactions
(Buchner & Walter, 2005, Ellis & Minton, 2006, Hartl, 1996).
The heat shock proteins, or hsps, are a large and diverse category of
molecular chaperones whose expression is enhanced during the conditions of
cellular stress. These proteins function to prevent protein aggregation during
stress conditions and increase the cell survival. However, it was subsequently
discovered that these proteins also have housekeeping functions, such as de
novo protein folding, resolubilization of protein aggregates, or protein degradation
((Buchner & Walter, 2005, Hartl, 1996, Hartl & Hayer-Hartl, 2002, Young et al,
2004). Besides hsps, numerous other chaperones had been discovered that
function in specific pathways and participate in a diverse range of activities, such
as assembly of oligomeric proteins or transport (Leroux, 2001). The general
function of these molecular chaperones is to bind to and stabilize unstable
conformers of their substrates in order to ensure their correct cellular fate (Hartl,
1996). Since chaperones are found in all three kingdoms of life and often are
essential for the cell survival, they are an important part of the proper functioning
of the cell (Leroux, 2001).
The three-dimensional structures of molecular chaperones provide
invaluable information on how these proteins are able to carry out their functions.
The structural basis of the chaperone-substrate interaction is especially
important, as it may reveal common strategies in the mechanism of the
chaperone action. This chapter discusses the structures of several molecular
chaperones with a special emphasis placed on the structural features that allow
them to interact with their substrates. The goal of this chapter is to identify
unifying schemes in the structures of molecular chaperones and the chaperone-
substrate interactions.
1.2. Trigger Factor
Trigger factor (TF) is the first chaperone to associate with the nascent
polypeptide just as it comes out of the ribosomal peptide exit tunnel (Hesterkamp
et al, 1996) and is found exclusively in eubacteria (Leroux, 2001). Trigger factor
associates with the large subunit of the bacterial ribosome in a 1:l ratio; the
association occurs through the ribosomal L23 protein located near the peptide
exit tunnel (Ferbitz et al, 2004, Kramer et al, 2002). TF works in cooperation with
other chaperones that associate with the newly synthesized polypeptide
downstream of TF, such as DnaK, GroEL, SecB, and the signal recognition
particle (SRP) (Deuerling et al, 1999, Ha et al, 2004, Kandror et al, 1995, Ullers
et al, 2006). TF and DnaK have overlapping substrate specificities (Deuerling et
al, 2003), and disruption of both DnaK and trigger factor genes causes massive
protein aggregation and is lethal to the cell (Deuerling et al, 1999). Unlike DnaK,
TF is ATP-independent and does not appear to be able to prevent the
aggregation of thermally denatured proteins in vitro (Schaffitzel et al, 2001). The
ribosome-bound TF prevents the proteinase K - induced degradation of large (up
to 41 kDa) stretches of unfolded polypeptides emerging from the ribosome
(Hoffmann et all 2006). The substrate binding specificity of TF was analyzed by
scanning cellulose-bound peptide libraries representing overlapping 13-mer
sequences of five Exoli proteins. TF prefers to bind eight residue stretches of
aromatic and basic amino acids with a positive net charge without positional
preference (Patzelt et al, 2001).
Several structures of TF are available, including a full-length structure
from Escherichia coli in complex with the ribosome (Ferbitz et all 2004), as well
as several other structures of the TF fragments alone or in complex with the
bacterial ribosomal proteins. The structure of TF is composed of three
functionally distinct domains: the N-terminal domain is responsible for the
interactions with the ribosome (Hesterkamp et al, 1996), and the middle domain
contains peptidyl-prolyl cisltrans isomerase (PPlase) activity (Maier et al, 2005).
The C-terminal domain has recently been demonstrated as the central module of
the chaperone activity (Merz et all 2006). Surprisingly, the three-dimensional
organization of these domains does not resemble the linear sequence, since the
C-terminal domain is located in the middle of the structure, between the N-
terminal and PPlase domains, which are located at the opposite ends of the
structure and do not interact with each other (Figure 1.1) (Ferbitz et all 2004,
Ludlam et all 2004). All interactions with the ribosome are accomplished by the
N-terminal domain through the conserved GFRxGxxP motif (Kramer et al, 2002)
located in the loop between the two a-helices (Ludlam et al, 2004).
The PPlase domain, which has been demonstrated as dispensable for the
chaperone activity (Kramer et al, 2004), resembles a well-described FKBP fold
found in several other PPlases (Ludlam et all 2004). The C-terminal domain is
composed of several a-helices that form two extended "arms" and is structurally
similar to the periplasmic chaperone SurA (Ferbitz et all 2004). TF is tethered at
the ribosome via its N-terminal domain, and the C-terminal domain is positioned
such that its "arms" hover over the ribosomal peptide exit side. The complete
Figure 1.1 The structures of the Trigger Factor, SurA, and MPN555.
A) A cartoon diagram of the Trigger Factor from Escherichia coli, PDB ID 1W26 (Ferbitz et al, 2004). The N-terminal domain is in green, the PPlase domain is in red, and the C-terminal domain is in blue. B) The protein surface electrostatics of Trigger Factor, PDB ID 1W26. Areas coloured white, red, and blue, correspond to neutral, negative, and positive surface electrostatics potential, respectively. Arrows indicate the location of the "arms" in the C-terminal substrate binding site C) Cartoon diagram of MPN555 from Mycoplasma pneumoniae (IZXJ, (Schulze-Gahmen et al, 2005)). D) Cartoon diagram of SurA from E.coli (lM5Y. (Bitto & McKay, 2002)). The PPlase domains are in red, and the substrate binding domain is in blue. E) The protein surface electrostatics of SurA. The peptide from a symmetry related molecule is shown as green cartoon. Arrows indicate the "arms" of the substrate binding domain. F) A side view of the substrate binding domain in (E), showing the peptide binding into the groove between the two "arms". The figures were made using PyMol (DeLano, 2002).
structure adopts a "crouching dragon" conformation, with a large "cradle"
between the N-terminal domain and the C-terminal "arms", which can
accommodate folded protein domains as large as 14 kDa (Ferbitz et all 2004).
The interior of the "cradle" is very hydrophobic. It has been suggested that this
"cradle" shields the newly synthesized polypeptides from the environment and
delays folding until a sufficient stretch of protein sequence is synthesized for a
protein domain to fold correctly (Ferbitz et al, 2004).
1.3. SurA and MPN555
SurA is a periplasmic, ATP-independent molecular chaperone that
promotes the folding and maturation of outer membrane porins, such as LamB,
OmpF, and OmpA (Behrens et all 2001, Hennecke et all 2005). SurA is selective
for its substrates, and prefers to bind to sequences enriched in aromatic residues
(Hennecke et al, 2005). Like trigger factor, SurA exhibits modular architecture.
SurA contains two FKBP-like PPlase domains, both of which are dispensable for
its chaperone activity, and a bipartite core domain composed of N- and C-
terminal sequences, which is responsible for the chaperone activity (Behrens et
al, 2001, Bitto & McKay, 2002). The catalytic domain P2 is located away from the
rest of the structure and is tethered to the rest of the protein via linkers, whereas
the second PPlase domain, PI , which is not catalytically active on its own, is
located close to the chaperone core domain (Figure 1.1) (Bitto & McKay, 2002,
Bitto & McKay, 2002). The substrate-binding domain is similar in fold to the C-
terminal domain of the trigger factor and to MPN555, a protein of unknown
function from Mycoplasma pneumoniae (Figure 1.1) (Schulze-Gahmen et al,
2005). It is composed of a helices which form two arm-like projections with a 50
14 hydrophobic crevice in between (Bitto & McKay, 2002). In the crystal structure
of SurA published by Bitto et a/, this hydrophobic crevice contains a peptide from
a neighbor molecule, which identifies it as a site of substrate binding (Figure 1 .I).
The peptide binds in an a-helical conformation between the arms of the core
domain. The inner surfaces of these arms contain hydrophobic pockets, which
interact with Leu153 and Val157 residues on the peptide. There are patches of
negative charge in the bottom of the crevice and on the inner surface of the
arms, which may provide potential for selecting specific substrates. The bound
peptide is only 15 14 long, but the crevice can potentially accommodate longer
peptides (Bitto & McKay, 2002).
1.4. Hsp7O and Hsp40
Hsp70 is the central component of the cellular system of molecular
chaperones; its homologues are found in almost all organisms, including
eubacteria, most archaea, and the cytosol and organelles of eukaryotes. The
general functions of this ATP-dependent chaperone include de novo folding of
polypeptides, prevention of protein aggregation, and refolding of aggregated
proteins (Mayer & Bukau, 2005a). In addition to playing a key role in the heat
shock response, hsp70 acts as a housekeeping protein during non-stress
conditions and participates in a wide variety of cellular processes, such as signal
transduction, protein translocation across membranes, protein quality control,
and interaction with regulatory proteins (Mayer & Bukau, 1998). The bacterial
hsp70 homolog DnaK has been estimated to assist the de novo folding of 10-
20% of newly synthesized polypeptides (Hartl & Hayer-Hartl, 2002). Most cells
encode multiple homologues of Hsp70 that are conserved in sequence yet carry
out diverse cellular roles. Almost all Hsp70 proteins interact with a J-domain co-
chaperone, such as hsp40 in the eukaryotic cytosol or DnaJ in E.coli, to stimulate
their intrinsically low ATPase activity and regulate interactions with substrates
(Hennessy et al, 2005). The J-domain, a highly conserved structure found at the
N-terminus of hsp40, is required for interaction with hsp70, however, many hsp40
proteins contain additional domains that allow them to carry out other specific
tasks (Qiu et all 2006). For example, some hsp40 proteins have their own
chaperone activity and form transient, ATP-independent associations with the
client proteins (Qiu et all 2006). In E.coli, which contains three hsp70 homologs
and six hsp40 homologs, there is a specific pattern of hsp701hsp40 interactions
whereby a particular hsp70 protein may have one or multiple hsp40 partners (Qiu
et all 2006).
Hsp70*ATP has low affinity and fast exchange rates for substrates,
whereas Hsp70*ADP has high affinity and slow exchange rates (Laufen et al,
1999). The following reaction cycle has been suggested for the E.coli hsp70
(DnaK). The substrate bound to DnaJ (an hsp40 homolog) is transferred to
DnaK*ATP. DnaJ also modulates the communication between the ATPase and
the substrate-binding domains of DnaK. Both DnaJ and substrate binding
stimulate the ATP hydrolysis by hsp70, which locks the substrate into the binding
cavity of hsp70. The substrate dissociates from hsp70 upon exchange of ADP for
ATP (Laufen et al, 1999, Young et all 2004).
In addition to hsp40, hsp70 recruits other proteins, such as nucleotide
exchange factors GrpE (in E.coli) and BAG-1 (in eukaryotes), as well as Hip,
which is thought to stabilize the ADP-bound state of hsp70; Hop, an hsp70-hsp90
organizing protein; and CHIP, which may connect hsp70 to the protein
degradation pathway (Mayer & Bukau, 2005b).
Hsp70 consists of a 45 kDa N-terminal ATPase domain (NBD) and a 25
kDa substrate binding domain (SBD) (Figure 1.2) (Mayer & Bukau, 2005a). The
ATPase and the substrate binding domains are connected by a linker of -10
residues long that may be responsible for the interdomain communication, which
couples substrate binding to ATP hydrolysis (Vogel et al, 2006). Numerous
structures of the separate ATPase and substrate binding domains of hsp70 are
available, as well as the structure of a functionally intact, nearly full-length bovine
hsc70 (a constitutively induced hsp70 homolog) that further highlights the role of
the interdomain linker in the communication between NBD and SBD (Jiang et all
2005). The N-terminal ATPase domain consists of two subdomains which form
two lobes with a deep cleft between them wherein the nucleotide binds (Flaherty
et al, 1990, O'Brien et all 1996). The substrate binding domain can be further
subdivided into a P-sandwich subdomain and a C-terminal a-helical subdomain
(Zhu et al, 1996) The structure of the SBD from E.coli DnaK in complex with a
synthetic peptide NRLLLTG reveals that the peptide binds only to the P-sandwich
subdomain, whereas the a-helical subdomain serves as a lid that locks the
substrate into place (Figure 1.2) (Zhu et al, 1996). The peptide binds in an
extended conformation and contacts DnaK through side-chain mediated van der
biz-
&-*- I-'
Figure 1.2 The structures of Hsp70 and Hsp40.
A) A cartoon diagram of bovine hsc70, an hsp70 hornologue. The nucleotide binding domains of hsc70 from PDB entries IYUW (Jiang et al, 2005) (in olive) and IKAX (O'Brien et al, 1996) (in purple) were superimposed with the r.m.s.d. of 1.38 A over 378 C, atoms. The program Superpose (Maiti et al, 2004) was used for superposition. ATP from entry IKAX is shown as orange spheres. The SBD from PDB entry 1YUW is in blue and lacks the 100 C-terminal residues. B) The substrate binding domain (blue, cartoon)of E.coli DnaK in complex with a peptide (green, spheres); PDB ID IDKX (Zhu et al, 1996). C) Left: as in B), except the SBD is coloured according protein surface electrostatics, and the peptide is shown as sticks. Right: a close-up view of the area bounded by a box in the left pane. The leucine binding pocket is indicated by an arrow. D) Left: A cartoon diagram of the C- terminal fragment of Ydjl, a Saccharomices cerevisae hsp40 homologue (green) in complex with a peptide (magenta, sticks). PDB ID INLT (Li et al, 2003). Right: a close-up view of the area bounded by a box in the left pane. The leucine binding pocket is indicated by an arrow. The surface of Ydj is coloured according to protein surface electrostatics, and the peptide is shown as sticks.
Waals interactions and main-chain hydrogen bonds. (Figure 1.2BC). A deep
hydrophobic pocket in the DnaK substrate binding site buries a leucine side chain
from the peptide and is important for substrate specificity. The dimensions and
biochemical characteristics of this pocket are ideal to bind leucine, but it may also
accommodate methionine, isoleucine, or smaller side chains. Bulky hydrophobic,
charged, or polar residues would be disfavoured at this position (Zhu et al, 1996).
The substrate binding site on DnaK is generally hydrophobic in nature, and has
an area of a slightly negative electrostatic potential adjacent to it (Zhu et al,
1996). This is consistent with the fact that DnaK prefers to bind stretches of four
to five hydrophobic residues flanked by basic residues, whereas acidic residues
are disfavoured (Rudiger et al, 1997).
The structure of the substrate binding domain of yeast hsp40 in complex
with a peptide substrate has been reported by Li et a1 (Figure 1.3) (Li et al, 2003).
The domain responsible for peptide binding consists of two P-sheets connected
by a short helix. The peptide GWYLEIS binds into an open hydrophobic groove
on the hsp40 surface by complementing one of the P-sheets as an antiparallel P-
strand. The chaperone-substrate interactions are similar to that seen in DnaK,
since they involve the peptide binding in an extended conformation via main-
chain hydrogen bonds and van der Waals contacts (Figure 1.2D). Importantly, a
central leucine residue on the peptide is buried in a hydrophobic pocket of hsp40,
analogous to that seen in the structure of hsp70 in complex with peptide (Li et al,
2003). This correlates well with the fact that hsp40 transfers the bound
substrates to hsp70, which would require the ability to bind to the same
substrates.
1.5. Hsp9O
The 90 kDa heat shock protein (hsp90) is a highly conserved and
abundant protein found in the cytosol of eubacteria and in the cytosol,
mitochondria, ER, and chloroplasts of eukaryotic cells (Picard, 2002). It is
essential in eukaryotes, and participates in folding and stabilization of specific
client proteins involved in a multitude of cellular processes, such as signal
transduction, transport, transcription, cell cycle regulation, and protein quality
control (Zhao & Houry, 2007). In cooperation with hsp70140, hsp90 may act in
refolding of misfolded proteins during stress conditions, however, it has not been
implicated in de novo protein folding (Mayer & Bukau, 1999). Hsp9O is ATP-
dependent and relies on various co-chaperones to regulate its ATPase cycle and
interactions with substrates (Pearl & Prodromou, 2006). For example, hsp90
cooperates with hsp70140 through an adaptor protein Hop to assist in folding of
steroid hormone receptors (Hernandez et all 2002), whereas the co-chaperone
cdc37 (p50) is recruited for interactions with the protein kinase clients (Roe et all
2004).
There are numerous structures of hsp90 available in the Protein Data
Bank (Berman et all 2000), including complexes with inhibitors and co-
chaperones. The overall structure of Hsp9O is modular and consists of three
domains arranged in a linear fashion: the N-terminal domain is responsible for
ATP and drug binding, the middle domain has been implicated in substrate
12
Figure 1.3 The structures of Hsp9O.
A) The closed conformation of Hsp9O. A cartoon diagram of Saccharomices cerevisae Hsp82, an Hsp9O homologue, in complex with ATP (2CG9, (Ali et al, 2006)). B) The open conformation of Hsp9O. A cartoon diagram of the nucleotide- free E.coli HtpG, an Hsp9O homologue (210Q, (Shiau et al, 2006)). In both A) and B), the N-terminal nucleotide binding domains are in green, the middle domains are in blue, and the C-terminal dirnerization domains are in red. ATP bound to the N- terminal domain in A) is in purple.
binding as well as contributing to the ATPase activity through a key catalytic
arginine residue, and the C-terminal domain is essential for Hsp9O dimerization
(Figure 1.3) (Pearl & Prodromou, 2006).
Two full-length hsp90 structures have been published recently: the yeast
hsp90 in complex with an ATP analogue and a co-chaperone p23lSbal (Ali et al,
2006), as well as an E.coli hsp90 homolog, HtpG, alone or in complex with ADP
(Shiau et al, 2006). These structures represent the three different nucleotide-
induced conformations of hsp90. In the nucleotide-free state, the dimeric hsp90
adopts an open, highly flexible V-shaped conformation. The two monomers
dimerize at the C-terminal domains via a four-helix bundle; a large hydrophobic
cleft between the two monomers is proposed to be the site of the substrate
binding (Figure 1.3B) (Shiau et al, 2006). Upon nucleotide binding, hsp90
undergoes significant conformational rearrangements and assumes a "closed"
state via the dimerization of the N-terminal domains (Figure 1.2A) (Ali et al,
2006). In the hsp90 structure in complex with a non-hydrolysable ATP analogue,
the two N-terminal domains form a stable dimer via the exchange of N-terminal
P-strands and hydrophobic interactions, and the middle domains are brought
close together (Ali et al, 2006). Although it has been proposed earlier that hsp90
encloses the client proteins in its central cleft acting as a "molecular clamp"
(Meyer et al, 2003b) , Ali et a1 demonstrated that in the ATP-bound "closed"
state, the dimeric cleft becomes too narrow to accommodate a folded substrate
(Ali et al, 2006). At present, it is not clear exactly how hsp90 binds its substrates,
necessitating further studies and perhaps determining the structure of hsp90 in
complex with a client protein to address this question.
1.6. Prefoldin (GimC)
Prefoldin is a heterohexameric chaperone found in archaea and in
eukaryotic cytosol, but not in eubacteria. Prefoldin binds to non-native proteins in
an ATP-independent manner and prevents them from aggregation. Prefoldin then
transfers its substrate to the class II chaperonin (eukaryotic CCT or archaeal
thermosome), thus acting as a co-chaperonin (Okochi et al, 2004, Vainberg et al,
1998). In eukaryotes, prefoldin seems to be specialized for binding cytoskeletal
proteins actin and tubulin, whereas in archaea, which lacks actin and tubulin, it
may play a more general role in protein folding similar to that of hsp7O (Leroux,
Figure 1.4 The structures of prefoldin and Skp.
A), B), C), and F) are based on the structure of archaeal prefoldin from Methanobacterium thermoautotrophicum. PDB ID 1FXK (Siegert et al, 2000). A) A cartoon diagram of the a-class prefoldin subunit. B) P-class prefoldin subunit. C) The prefoldin hexamer, side view. a-subunits (orange) are located in the middle of the structure and serve as the central points for oligomerization of the P-subunits (green). D) and E) are based on the structure of E.coli Skp, PDB ID 1SG2 (Korndorfer et al, 2004). D) A monomer of Skp. E) Skp trimer, side view. F) Surface representation of prefoldin, view from the bottom, coloured according to the surface vacuum electrostatics.
2001). The prefoldin binding site in actin and tubulin is signified by the motif
EHGl preceded by several hydrophobic residues (Rommelaere et all 2001).
The prefoldin hexamer is composed of two types of prefoldin subunits:
class a and class p. Archaea possess only one homolog of each class, whereas
eukaryotes have two class a homologs and four class P homologs (Siegert et al,
2000). The crystal structure of the archaeal prefoldin was solved by Siegert et a1
in 2000. The individual structures of the a- and P-class subunits are similar in that
they have two long a-helices at the N-terminus and C-terminus, which fold over
to make a coiled coil. The central part of each subunit contains one (in p-class) or
two (in a-class) P-hairpin structures, which are responsible for oligomerization by
forming a central platform of two P-barrels (Figure 1.4). The overall structure of
the assembled prefoldin hexamer resembles a jellyfish with the central P-barrels
serving as the "body" from which six flexible coiled coil "tentacles" extend (Figure
1.4) (Siegert et al, 2000). The coiled coils are partially untwisted at their tips and
expose hydrophobic patches that are crucial for interaction with the substrates
(Okochi et all 2004, Siegert et al, 2000). An additional region of hydrophobicity is
located at the bottom of the cavity formed by the six prefoldin coiled coil
"tentacles" and has been proposed to protect the unfolded substrates from the
cytosolic environment (Siegert et al, 2000). The complex between eukaryotic
prefoldin and actin as revealed by electron microscopy shows an unfolded actin
binding into the central cavity of prefoldin and interacting with the tentacle tips
(Martin-Benito et all 2002). In addition, prefoldin was shown to bind to the apical
domains of one or both CCT rings via the tips of its tentacles (Martin-Benito et al,
2002). In prefoldin, the binding sites for the chaperonin appear to be adjacent to
the peptide-binding sites, which may be important for efficient substrate transfer
(Okochi et all 2004).
1.7. Skp
Skp is a periplasmic molecular chaperone of Gram-negative bacteria that
is involved in biogenesis of outer membrane porins, such as OmpA, OmpF,
OmpC, and LamB (Chen & Henning, 1996). The proteins that are being
translocated across the inner membrane in an unfolded state are prone to
aggregation immediately upon emergence in the periplasm. Skp binds to its
target proteins at the inner membrane as they emerge from the Sec translocon,
thereby protecting them from misfolding and aggregation in the periplasm (Harms
et all 2001). In addition, Skp facilitates the insertion of its substrates into the
outer membrane, a function that requires Skp interaction with a
lipopolysaccharide (Bulieris et all 2003). Two other periplasmic chaperones,
SurA and DegP, have been implicated in interactions with outer membrane
proteins. On the basis of the analysis of null mutations of these three
chaperones, it has been suggested the periplasm of E.coli contains two parallel
pathways for folding and insertion of outer membrane proteins, where Skp and
DegP are part of one pathway, and SurA is part of a separate pathway (Rizzitello
et all 2001).
Two crystal structures of E.coli Skp are currently available in the PDB
database. The structure of the monomeric Skp can be divided into two
subdomains: the core subdomain and the a-helical extensions from the core. The
17
core subdomain is composed of sequences located at the N- and C-termini and
contains two short a-helices and four @-strands (Walton & Sousa, 2004). This
core subdomain is responsible for the formation of the functional Skp
homotrimer. The middle part of each monomer contains two long a-helices joined
by a loop, which run along one another in an antiparallel fashion (Figure 1.4)
(Walton & Sousa, 2004). The core subdomains from each monomer associate
via their @-strands to form three inter-subunit @-sheets around the central 3-fold
axis. The a-helical subdomains are flexible and project away from the trimeric
core (Korndorfer et al, 2004). The inside surfaces of the a-helical "tentacles" are
hydrophobic in character and are arranged around the central cavity, which is a
plausible site for substrate binding (Korndorfer et al, 2004, Walton & Sousa,
2004). The tip of each "tentacle" contains a region of positive charge, which is
different from prefoldin, where the tentacle tips are hydrophobic and are involved
in substrate binding (Korndorfer et al, 2004).
Strikingly, the assembled structure of Skp resembles the jellyfish shape
with a-helical "tentacles", which is also seen in prefoldin, in spite of the different
topology of the secondary structural elements and no sequence similarity
between the two proteins. (Figure 1.4) (Korndorfer et all 2004). It is therefore
possible that the structural and functional similarities between prefoldin and Skp
arose via convergent evolution (Korndorfer et all 2004).
1.8. LolA
The periplasmic chaperone LolA is part of the E.coli Lol system
responsible for the sorting and localization of lipoproteins to the outer membrane.
18
Mature lipoproteins in the periplasm contain an N-terminal cysteine residue
modified with a lipid moiety (Tokuda & Matsuyama, 2004). Lipoproteins that are
destined to remain in the inner membrane contain an aspartic acid residue at
position 2, which serves as Lol avoidance signal (Tokuda & Matsuyama, 2004).
Other components of the Lol system include LolCDE, an ATP-binding cassette
(ABC) transporter anchored in the inner membrane, and LolB, a lipoprotein
receptor in the outer membrane. LolCDE releases the lipoproteins from the inner
membrane, transferring them to the chaperone LolA, which carries the
lipoproteins across the periplasmic space and transfers them to LolB in the outer
membrane. The lipoprotein transfer from LolA to LolB is ATP-independent and
occurs because LolB has higher affinity to lipoproteins than that of LolA (Tokuda
& Matsuyama, 2004).
The crystal structure of LolA from E.coli is available in the Protein Data
Bank (Takeda et al, 2003). LolA consists of an I I-stranded antiparallel P-sheet
forming un unclosed P-barrel, and 3 a-helices located on one face of the sheet
(Figure 1.5A). The inner surface of the P-sheet contains a hydrophobic cavity that
may act as a binding site for the lipid moieties on lipoproteins. The three a-
helices are also hydrophobic in character and act as a lid to the putative binding
site (Takeda et al, 2003). The lid is "locked" in place via interactions between
arginine 43 of the P-sheet and the residues in the a-helices, but is expected to
open and close upon binding and release of lipoproteins (Takeda et al, 2003). A
recent study which involved mutating the residues forming the hydrophobic cavity
and the a-helical lid demonstrated that both these regions are crucial for binding
Figure 1.5 The structures of LolA and LolB.
A) The structure of LolA chaperone (liwl, (Takeda et al, 2003)) in cartoon representation. B) The structure of LolB receptor in complex with PEG 2000 MME (IIWN, (Takeda et al, 2003)). LolB is shown as pink ribbon, and PEG2000 MME as green sticks.
lipoproteins and their transfer to LolB (Watanabe et al, 2006).
Interestingly, the structure of LolB receptor is strikingly similar to that of
LolA chaperone, despite the low sequence identity of 8%. In contrast to LolA,
however, the a-helical lid of LolB is in an open conformation, and the putative
binding site accommodates a molecule of polyethylene glycol 2000 monomethyl
ether (PEG 2000 MME), a compound used to crystallize the protein (Figure 1.5B)
(Takeda et all 2003)
1.9. PapD, FimC
Gram-negative pathogenic bacteria utilize thread-like extracellular
adhesive projections termed pili in order to recognize and interact with their
specific receptors on the surface of the host cells. Pili are important virulence
factors and are responsible for a wide variety of infectious diseases (Piatek et al,
2005). The E.coli Type 1 and P pili, as well as over 30 other pili and non-pili
structures from various Gram-negative pathogens, are assembled in the
periplasm via the chaperone-usher pathway (Sauer et all 2000). Both type 1 and
P pili have similar heteropolymeric structure, and are encoded by the E.coli fim
and pap gene clusters, respectively. They are rigid helical rods assembled from
identical pilin subunits, FimA in type 1 and PapA in P pili. Each rod is joined to a
thinner tip fibrillum, made up of other types of fim or pap subunits, followed by an
adhesin subunit at the distal end. Two proteins are responsible for the pili
assembly: a soluble periplasmic chaperone (FimC in type 1 and PapD in P pili)
and an usher (FimD in type 1 and PapC in P pili), integrated into the outer
membrane (Capitani et all 2006, Sauer et all 2000).
FimC and PapD chaperones are responsible for binding the fim or pap
type pilin subunits, respectively, as they emerge from the Sec translocon, and
targeting them to their respective ushers, which add the subunits to the growing
pilus fiber on the outer cell surface. The subunit structure consists of an
incomplete immunoglobulin-like (lg) fold, missing the seventh, C-terminal strand.
In the assembled pilus structure, this seventh strand is supplied by a highly
conserved N-terminal extension upstream of the lg fold in an adjacent subunit
(Sauer et al, 2002). The interactions of the pili subunits with the chaperone and
the usher proceed in an ATP-independent manner.
In the absence of the chaperone, the pilus subunits do no fold properly
and aggregate (Barnhart et all 2000). The chaperone binding stabilizes the
subunit and prevents it from aggregation or premature interactions with other pili
subunits (Kuehn et al, 1991). The structures of FimC and PapD chaperones are
very similar (Piatek et all 2005). The chaperones consist of two 19-like domains,
with each domain containing seven antiparallel P-strands divided into two P-
sheets that pack against one another. The two domains are arranged at an angle
to one another, such that the complete structure resembles a boomerang. The
two domains are joined by a -30 residue long linker, enriched in hydrophobic
amino acids (Holmgren & Branden, 1989).
Several structures of FimC and PapD chaperones in complex with their
respective pilus subunits have been obtained. These structures reveal the basis
of the chaperone-subunit interaction. Unlike many cytoplasmic chaperones, FimC
and PapD bind their substrates in a native-like state (Figure 1.6) (Kuehn et al,
1991). The chaperone stabilizes the subunit via the donor-strand
complementation mechanism, which involves donating one of its own strands to
complete the lg fold of the pilus subunit (Choudhury et all 1999, Sauer et al,
1999). In the PapD-PapK structure, the chaperone PapD inserts its strand G I
into a hydrophobic groove between the strands A and F of PapK (Figure 1.5AB).
The inserted strand makes parallel P-sheet interactions with the subunit strand F,
thus creating a non-canonical lg fold (Sauer et all 1999). This is in contrast to the
pili subunit-subunit complexes, where the N-terminal extension from one subunit
complements the F strand of another subunit in an antiparallel fashion, making a
more stable, canonical lg fold (Sauer et al, 2002). Since the pili subunit-subunit
interactions are more stable than that of chaperone-subunit, the displacement of
the chaperone G I strand in the subunit groove by the N-terminal extension of
Figure 1.6 The structures of the type 1 pili chaperone FimC and the P pili chaperone PapD.
A) and 6) are based on the structure of PapD in complex with a pilin subunit PapK (PDB ID IPDK (Sauer et al, 1999)). A) A cartoon diagram of PapD (green) and PapK (cyan). The PapD strand G I forms a parallel P-sheet with the PapK strand F. B) PapK is shown as surface coloured according to the electrostatic potential. The G I strand of PapD is shown as sticks. C) and D) are based on the structure of FimC in complex with the adhesin subunit FimH (IQUN (Choudhury et al, 1999)). The N- terminal lectin domain of FimH is not shown for clarity. C) A cartoon diagram of FimC (orange) and FimH (purple). D) FimH is shown as surface coloured according to the electrostatic potential. The GI strand of FimC is shown as sticks.
another subunit may be energetically favourable and may drive the fiber
formation (Sauer et all 2002).
The chaperone G1 strand has a pattern of alternating hydrophobic
residues, which is also found in the N-terminal extensions of the pilus subunits
(Sauer et all 1999). The G1 strand interacts with the residues of the groove via
main-chain hydrogen bonds to strands A and F, as well as hydrophobic
interactions with the base of the groove. In addition, there are several side-chain
mediated hydrogen bonds between the chaperone and the C-terminus of the
subunit that serve to anchor the subunit into the cleft between the two domains of
the chaperone (Sauer et all 1999). A very similar structure and the mechanism of
donor strand complementation were reported for the FimC-FimH complex (Figure
1.6CD) (Choudhury et all 1999).
Several roles were suggested for the chaperone in the pili biogenesis
pathway. First, the chaperone stabilizes the pili subunit and prevents it from
unproductive aggregations by capping the hydrophobic groove in the subunit
(Choudhury et all 1999, Sauer et all 1999). Second, it keeps the subunit primed
for displacement of the chaperone strand G1 by the N-terminal extension of
another subunit, necessary for assembly into the pilus fiber (Sauer et all 2002).
Third, it may facilitate the folding of the subunits in the periplasm, possibly by
providing the missing steric information necessary for correct folding of the
subunit (Barnhart et al, 2000, Vetsch et all 2004).
I . lo. Type Ill secretion chaperones
Many Gram-negative pathogenic bacteria interact with the host cells by
directly injecting their virulence factors into the host cytosol via the type Ill
secretion machine, a needle-like structure that is anchored in the inner
membrane of the pathogen and projects through the outer bacterial membrane
and the host membrane (Galan & Wolf-Watz, 2006). Within it, the needle
contains a narrow channel allowing a direct delivery of the virulence factors from
the bacterial cytoplasm into the host cytoplasm. In addition to the structural
proteins that make up the needle, each type Ill secretion system also encodes for
the transcriptional regulators, virulence factors (effectors and translocators), and
chaperones (Parsot et all 2003). The virulence factors are delivered into the host
cell cytosol and interfere with the host signaling (effectors) or make a pore in the
host cell membrane (translocators) (Feldman & Cornelis, 2003). Many effectors
and translocators interact with the chaperones, which stabilize them, prevent
their aggregation, target them to the secretion apparatus, and may establish an
order of secretion for the effectors (Feldman & Cornelis, 2003, Parsot et al,
2003). These chaperones are small, acidic, homodimeric proteins that
specifically recognize only one or two effectors (Galan & Wolf-Watz, 2006).
Although there is no sequence homology between the type Ill secretion
chaperones, their three dimensional structures are remarkably similar. Each
chaperone monomer consists of an antiparallel P-sheet composed of 5 P-strands,
and 3 a-helices, all located on the same side of the P-sheet. The helix a2 and
strand P4 interact with the symmetric elements in the other monomer, forming the
patch )d 1
zk. patch 2
Figure 1.7 The structures of the Type Ill secretion system chaperones in complex with their effector substrates.
Left panes show cartoon diagrams, with the chaperones in brown and the substrates in green. Right panes show the chaperones as surface coloured according to electrostatic potential, and the substrates as cartoon in green. A) SycE in complex with the chaperone binding domain of YopE ('IL2W, (Birtalan et al, 2002)). The hydrophobic patches 1 and 2 that interact with the substrates (Birtalan) are identified by arrows. B) SicP in complex with the chaperone binding domain of SptP (IJYO, (Stebbins & Galan, 2001)). C) The heterodirneric chaperone SycNNscB in complex with a nearly full length effector YopN. SycN is in brown, YscB is in red, and YopN is in green (IXKP, (Schubot et al, 2005)).
dimer interface (Figure 1.7) (Birtalan et al, 2002, Luo et al, 2001, Stebbins &
Galan, 2001, Yip et al, 2005).
Most effectors and translocators contain a secretion signal located within
the first 20-30 residues at the N-terminus, which appears unstructured and not
conserved in sequence. The stretch of -50-100 amino acids downstream of the
signal sequence typically participates in the interaction with the chaperone and is
called a chaperone binding domain (CBD) (Galan & Wolf-Watz, 2006). There are
several crystal structures of the chaperones bound to their effectors, which
highlight the basis of their interactions. From the crystal structure of the
chaperone SycE complexed with the CBD of its substrate effector YopE from
Yersenia pseudotuberculosis, it is clear that the CBD of YopE wraps around the
dimeric SycE (Figure 1.7A) (Birtalan et all 2002). In doing so, YopE forms
extensive surface contacts with both monomers of SycE and drapes around
more than half of the circumference of the chaperone. Most of the bound CBD
from YopE is in an unfolded, extended conformation, which makes contacts with
the surface of the chaperone through main chain hydrogen bonds and numerous
specific polar and non-polar interactions. Birtalan et a1 identifies two pairs of
symmetric hydrophobic patches in SycE that interact with the secondary
structural elements found in the CBD of YopE. Patch 1 makes contact with YopE
helices a1 and a2, whereas patch 2 interacts with the strands P I and P2 (Figure
1.7A) (Birtalan et all 2002). The comparison of the SycE structures with and
without the substrate bound indicate that the chaperone structure is static and is
not affected by the substrate binding (Birtalan et all 2002). The co-crystal
structure of the chaperone SicP and the effector SptP reveals a striking similarity
to the SycE-YopE complex. SptP wraps around its chaperone in essentially the
same way as YopE, although there is no sequence similarity between either the
chaperones or the effectors (Figure 1.7B) (Stebbins & Galan, 2001).
Even though the structures described above demonstrate that the
chaperone binding domain of the effector binds to its cognate chaperone in an
unfolded form, several studies indicate that the chaperone-bound effectors still
retain their activity. It was therefore concluded that the chaperone does not
unfold the entire effector and only interacts with its CBD (Akeda & Galan, 2005,
Birtalan et al, 2002). This notion was confirmed by the co-crystal structure of the
heterodimeric chaperone SycN-YscB and its effector substrate YopN, crystallized
in nearly full length (Figure 1.7C) (Schubot et all 2005). Apart from the CBD,
which forms extensive interactions with the chaperone similar to those previously
described for SycE-YopE and SicP-SptP complexes, the rest of the effector
forms a folded domain whose conformation does not seem to be affected by the
chaperone binding (Schubot et al, 2005). CBD is required for the subsequent
interaction of the effector with a highly conserved, membrane associated
ATPase, which unfolds the entire effector prior to its secretion through the needle
apparatus (Akeda & Galan, 2005). This suggests that the chaperone masks the
aggregation-prone CBD and delivers the effector to the peripheral ATPase, which
then displaces the chaperone and unfolds the effector (Akeda & Galan, 2005,
Letzelter et al, 2006).
1 .I 1. Signal Recognition Particle
The signal recognition particle (SRP) is one of the chaperone components
of the Sec-dependent protein translocation system and is universally conserved
28
across the three kingdoms of life (Driessen et all 2001). SRP recognizes the
hydrophobic portion of the N-terminal signal peptide or internal signal anchor
sequence on the proteins that are destined for secretion or insertion into the
membrane (Fekkes & Driessen, 1999). SRP acts co-translationally, binding the
ribosome - nascent protein complex (RNC), and delivering it to the plasma
membrane (in prokaryotes) or to the endoplasmic reticulum (in eukaryotes). SRP
efficiently competes with the trigger factor for binding nascent chains at the
ribosome when a signal sequence of sufficient hydrophobicity is synthesized
(Ullers et all 2006). Eukaryotic SRP causes an arrest in further translation by the
bound ribosome (Halic & Beckmann, 2005). At the membrane, SRP-RNC
complex interacts with the SRP receptor (SR). This interaction is mediated by
GTPases that are present in both SRP and SR and activate each other in a
reciprocal fashion, which leads to docking of the RNC to the membrane and
transfer of the bound polypeptide to the Sec translocon (Egea et al, 2005, Halic &
Beckmann, 2005).
SRP is a ribonucleoprotein, which, in mammals, consists of six proteins
(SRP54, 19, 68, 72, 9, 14), and a 300-nucleotide long 7s RNA molecule. SRP
can be subdivided into two domains. The Alu domain, which is responsible for
the elongation arrest, consists of SRP9 and 14 and 3' and 5' ends of the RNA.
The S domain, necessary for signal sequence recognition and interaction with
SR, contains the remainder of the proteins and the majority of the RNA (Halic &
Beckmann, 2005). In archaea, SRP is less complex and contains only SRP54
and 19, as well as the 7s RNA similar to that found in eukaryotes. Eubacteria
il Finger loor,
I,,
Finger h
Figure 1.8 The structures of the Signal Recognition Particle.
A) A cartoon diagram of Thermus aquaticus Ffh with a peptide from a symmetry related molecule in the substrate binding groove. The NG domain is in olive, the M- domain is in red, and the peptide is in green (2FFH, (Keenan et al, 1998)) 6) Same as in A), except Ffh is shown as surface coloured according to electrostatic potential. C) A cartoon diagram of the superposition of the M-domains from structures with (2FFH) and without (lQZX, (Rosendal et al, 2003)) the bound peptide. Note the variation in position of the finger loop. D) A complex between the ribosome (green surface), eukaryotic SRP (purple cartoon) and a signal sequence peptide (red cartoon) PDB ID 2J37 (Halic et al, 2006).
contain an even simpler version of SRP, with only one protein Ffh, a homolog of
SRP54, and a 110-nucleotide long 4.5s RNA (Egea et al, 2005, Luirink &
Sinning, 2004).
In the structure of the E.coli SRP in complex with the RNC, obtained by
electron microscopy, SRP contacts the ribosomal protein L23 in the absence of
the signal peptide, and forms contacts at three other sites on the ribosome in its
presence (Schaffitzel et al, 2006). This allows SRP to scan the nascent peptide
and form full contact with the ribosome once the signal sequence is found. SRP
positions itself at the peptide exit tunnel on the ribosome, but does not cover the
peptide completely, which provides the opportunity for other factors to bind
(Schaffitzel et al, 2006).
The protein SRP54 (Ffh) and the RNA helix 8 (domain IV in eubacteria)
comprise the SRP core, which is highly conserved in all species (Luirink &
Sinning, 2004). SRP54 consists of the N-terminal NG-domain, which contains the
GTPase activity and is required for the interaction with the SRP receptor at the
membrane, and a C-terminal methionine-rich M-domain, which mediates binding
to the signal peptide and interaction with the SRP RNA (Figure 1.7) (Luirink &
Sinning, 2004). The NG and M domains are joined by a flexible linker, and the
whole molecule adopts an L-shape (Figure 1.8) (Rosendal et al, 2003).
The structure of the M-domain of SRP54 is composed of several
amphipathic a-helices that form a deep hydrophobic groove, which likely serves
as a site of the signal peptide binding. The inner lining of the peptide-binding
groove is rich in methionines, a feature that is conserved in all SRP54 (Keenan et
al, 1998, Rosendal et all 2003). Since the methionine side chain is hydrophobic
and conformationally flexible due to lack of branching, it was proposed that this
feature was an adaptation to accommodate a variety of hydrophobic residues
found in signal peptides (Bernstein et al, 1989). The N-terminal helices aM1 and
aM2 are connected by a long "finger loop" that might act like a lid to the peptide
binding site (Keenan et al, 1998, Rosendal et al, 2003). In the Thermus aquaticus
structure of the M-domain, the peptide-binding groove is occupied by a portion of
a symmetry related molecule, and thus the "finger-loop" lid is stabilized in an
"open" conformation (Figure 1.8AB) (Keenan et al, 1998). In the crystal structure
of the M-domain from Sulfolobus solfataricus, there is no peptide occupying the
hydrophobic groove, and the "finger loop" lid adopts a "closed" conformation,
folding over the C-terminal helix aM5 and covering the peptide binding groove,
which presumably stabilizes and protects it from the solvent (Figure 1.8C)
(Rosendal et all 2003). This highlights the functional role of the flexibility of the
"finger loop" lid. The closing of the "finger loop" lid causes movement of the
adjacent a-helices and rearrangement of the N-terminal part of the M-domain.
The C-terminal part of the M-domain remains stable. It contains an arginine-rich
helix-turn-helix motif that is involved in binding of SRP RNA (Rosendal et all
2003).
The recently published structures of E.coli SRP-70s RNC and mammalian
SRP-80s RNC complexes obtained by cryo-EM show that the M-domain of
SRP54 is located adjacent to the ribosomal peptide exit tunnel and the
hydrophobic groove of the M-domain is occupied by the density that corresponds
to the signal sequence (Figure 1.8D) (Halic et al, 2006).
I .12. SecB and CsaA
SecB is another chaperone involved in the Sec-dependent protein
secretion, but unlike SRP, it acts post-translationally. SecB binds to newly
synthesized pre-proteins in a non-native conformation and prevents their folding
and aggregation while delivering them to SecA, a peripherally bound component
of the Sec translocon at the inner membrane. The SecA ATPase then mediates
the insertion of the pre-protein into the SecYEG translocation channel (Driessen
et all 2001, Randall & Hardy, 2002, Zhou & Xu, 2005). SecB is only found in
Gram-negative eubacteria, and its substrates are secretory and outer membrane
proteins (Baars et all 2006). SecB does not use the N-terminal signal peptide to
recognize its substrates and binds instead to a motif of nine residues of basic
and aromatic amino acids (Knoblauch et all 1999). The secretory pre-proteins
destined for export via the SecB pathway are first recognized by the trigger
factor, which prevents the pre-protein association with SRP and enables their
interaction with SecB (Beck et all 2000, Mitra et all 2006).
CsaA is another protein that has been demonstrated to act as a
chaperone in the Sec-dependent protein secretion pathway. CsaA was first
identified in Bacillus subtilis, which lacks SecB, and was shown to bind SecA and
several secreted precursors and to prevent the protein aggregation (Linde et al,
2003, Muller et all 2000a, Muller et al, 2000b). CsaA is present in certain families
of Gram-positive eubacteria, as well as in some Gram-negative eubacteria and
archaea. CsaA prefers to bind to peptide stretches with a positive net charge,
which contain hydrophobic residues flanked by basic residues. These sequences
are likely to be found in the folded core of mature proteins, but not in the signal
peptides (Linde et al, 2003).
SecB and CsaA share no sequence or structural homology. SecB is a
homotetramer, in which each monomer is composed of a 4-stranded antiparallel
P-sheet and two a-helices located on the same side of the P-sheet. The
tetrameric molecule is organized as a dimer of dimers. First, the P-strands of the
two monomers associate to form a combined eight-stranded antiparallel P-sheet,
with the a-helices beneath it. Then, the two dimers associate via their a-helices in
such a way that the a-helices are sandwiched between the two P-sheets (Dekker
et al, 2003, Xu et al, 2000). Two long hydrophobic grooves on the interface
between the two P-sheets are present on either side of SecB, and were proposed
to be the sites of the substrate binding (Figure 1.9A) (Dekker et al, 2003, Xu et al,
2000). Xu et a1 subdivides the proposed substrate binding groove into two
subsites made up of well conserved residues: a deep subsite 1, lined with
aromatic residues, and a shallow subsite 2, lined with hydrophobic residues (Xu
et al, 2000). In addition, the substrate binding groove is rimmed with negatively
charged residues (Figure 1.9B). Overall, the biochemical nature of the substrate
binding groove correlates well with the preference of SecB to bind peptides
enriched in hydrophobic and basic residues (Xu et al, 2000). A recent study
maps the binding interface between SecB and its unfolded substrate by site-
directed spin labelling and reveals that the substrate wraps around the SecB
chaperone and binds into the grooves identified by Xu et a1 (Crane et al, 2006).
In addition to these grooves, the substrate might take several possible routes
Figure 1.9 The structures of SecB and CsaA.
A) A cartoon diagram of the homotetrameric SecB ('IFX3, (Xu et al, 2000)). B) A surface representation of SecB (1FX3). Left pane: the putative substrate binding subsites 1 and 2 are coloured green and magenta, respectively. Right pane: SecB surface coloured according to vacuum surface electrostatics. C) The structure of CsaA (2NZ0, (Shapova & Paetzel, 2007)). Left pane: a cartoon diagram of the homodimeric CsaA. Right pane: a surface representation, coloured according to protein electrostatics. The location of the putative substrate binding site is indicated by an arrow.
around the chaperone forming contacts with hydrophobic, polar, and charged
residues (Crane et al, 2006).
In contrast to SecB, CsaA is a dimer, with each monomer composed of a
5-stranded P-barrel with a short capping a-helix reminiscent of an
oligonucleotide-oligosaccharide binding (OB) fold. Short N- and C-terminal
extensions from the central P-barrel form the dimer interface. There are two large
hydrophobic cavities located at the side of each P-barrel that were proposed to
act as substrate binding sites (Figure 1.9C) (Kawaguchi et al, 2001, Shapova &
Paetzel, 2007). The entrance to each cavity contains a cluster of the negative
electrostatic surface potential, which is consistent with the fact that CsaA prefers
to bind peptides with a positive net charge. The substrate might then wrap
around the surface of CsaA in the same way as was reported for SecB (Shapova
& Paetzel, 2007).
1.13. TorD
The twin-arginine (Tat) protein transport system which exists in
eubacteria, archaea, and eukaryotic chloroplasts is dedicated to the transport of
fully folded proteins across the membrane (Lee et al, 2006, Palmer et al, 2005).
Tat substrates are respiratory enzymes that contain redox-active co-factors. They
require assembly before translocation across the membrane and therefore are
incompatible with the Sec translocation system which transports its substrates in
an unfolded form. Tat substrates are synthesized with an N-terminal signal
sequence containing SRRxFLK "twin-arginine" motif. The Tat system chaperones
were proposed to bind and "mask" this signal sequence to prevent premature
36
targeting of unassembled Tat substrates to the Tat translocon (Lee et al, 2006,
Palmer et al, 2005). They also play a role in the assembly of the substrates and
insertion of the co-factors (Lee et al, 2006).
Figure 1.10 The structure of TorD.
A cartoon representation of dimeric TorD (INIC, (Tranier et al, 2003)). Subunits A and B are shown in pink and blue, respectively.
TorD is a specific chaperone for TorA molybdoenzyme, a periplasmic
triethylamine N-oxide (TMAO) reductase. The structure of dimeric TorD from
Schewanella massilia was determined at 2.4 A resolution (Tranier et al, 2003).
TorD from this bacterium forms multiple oligomeric species; the monomeric and
the dimeric form can both facilitate assembly of TorA (Tranier et al, 2003). TorD
is made up entirely of a-helices, and the two monomers exhibit domain
swapping. Each monomer contains 10 a-helices that can be subdivided into two
domains that are linked by a hinge region. The N-terminal domain of one
monomer (6 helices) interacts with the C-terminal domain of the other monomer
(4 helices) such that the entire structure of the dimer resembles a "dumbbell"
shape with two distinct "lobes" (Figure 1 . lo) (Tranier et al, 2003). This structure,
however, may represent a folding intermediate rather than the true biological fold
of TorD, as it does not appear thermodynamically stable due to exposure of
several hydrophobic residues to the solvent (Tranier et al, 2003). The authors
suggest that each "lobe" within the structure of dimeric TorD could exist as an
independent entity formed by a single TorD monomer. Instead of domain
swapping, which requires the hinge region to be in an extended conformation,
this region could form a loop, bringing the C-terminal domain back to interact with
the N-terminal domain of the same monomer (Tranier et all 2003).
Several other Tat chaperones are known, such as DmsD, NarJ, YcdY,
HyaE, and HybE. The structures of these proteins are not yet available, and
solving them would help determine the mechanism of Tat chaperone-substrate
interaction in greater detail.
I .l4. GroEL and GroES
GroEL and its co-chaperonin GroES, also known as hsp60 and hspl0,
respectively, belong to a Group I family of chaperonins, which are found in
eubacteria and in eukaryotic mitochondria and chloroplasts. GroEL is essential in
E.coli, and although it does not bind nascent proteins, it acts downstream of the
trigger factor and DnaK and assists the folding of an estimated 10% of cytosolic
proteins (Hartl & Hayer-Hartl, 2002). Approximately 85 cytosolic proteins exhibit
an obligatory dependence on the GroEL system to reach their native state.
These are large proteins with complex topologies that fold slowly and tend to
aggregate due to prolonged exposure of hydrophobic residues during folding
(Kerner et al, 2005).
GroEL is a barrel-like assembly of 14 identical 57 kDa monomers
arranged in two heptameric stacked rings (Figure 1.9). Each ring contains a 45
&wide central cavity that is separated from the cavity in the other ring. Each
monomer consists of three domains: the apical domain that binds unfolded
substrates and GroES, the equatorial domain that binds ATP and is involved in
the interactions within and between the two rings, and a flexible intermediate
domain that connects the two other domains (Figure 1.1 1A). In the assembled
GroEL structure, the two rings are arranged such that the equatorial domains of
their subunits face each other (Figure 1.11B) (Bartolucci et al, 2005, Braig et al,
1995). The interior of the apical domains is lined with hydrophobic amino acids,
which bind the exposed hydrophobic residues on the substrates (Fenton et all
1994).
GroES is a heptameric, dome-shaped ring approximately 75 A in
diameter, composed of -10 kDa subunits arranged with a 7-fold symmetry (Hunt
et al, 1996). GroES binds to the cis ring of GroEL in the presence of ATP or ADP,
acting like a lid to cover the central chamber of GroEL (Figure 1.11C). The
interaction with GroEL occurs via a "mobile loop" segment that swings away from
the p-core of each GroES subunit and binds the apical domain on the
corresponding GroEL subunit (Xu et al, 1997). The crystal structure of the apical
domain of GroEL in complex with a phage display-selected peptide revealed that
the substrate binds into a hydrophobic groove between two a-helices, which has
also been implicated of binding the GroES mobile loop (Figure 1 .I ID) (Chen &
Sigler, 1999). These substrate binding grooves from the seven subunits of a
GroEL ring face the interior of the central GroEL cavity and surround it like an
adhesive ring that likely serves to capture parts of an unfolded polypeptide (Chen
& Sigler, 1999).
Figure 1.11 The structures of GroES and GroEL.
A) and B) The structure of apo-GroEL from E.coli (IXCK (Bartolucci et al, 2005)). A) A cartoon representation of GroEL monomer. Apical domain is in blue, intermediate domain is in red, and equatorial domain is in green. B) A surface representation of the GroEL tetradecamer. Two monomers in each ring are coloured according to the colouring scheme in A). C) A cartoon representation of the complex between E.coli GroEL and GroES (IAON, (Xu et al, 1997)). GroES is in purple, the cis ring of GroEL is in orange, and the trans ring is in green. D) A complex between the apical domain of GroEL (blue) and a phage-display derived peptide (green). PDB ID 1 DKD (Chen & Sigler, 1999). Left pane: a cartoon representation. Right pane: GroEL is shown as surface coloured according to electrostatic potential.
GroEL is a functionally asymmetric molecule; only one of its rings (the cis
ring) is active at a time, but this role is alternated between the two rings in a cycle
controlled by ATP binding and hydrolysis. The binding of GroES and the
nucleotide brings about dramatic rearrangements in the cis ring (Xu et al, 1997).
First, the movement of the intermediate domain closes the nucleotide-binding
sites in the equatorial domain so that the free dissociation of ADP from the cis
ring is impeded. Second, the apical domains undergo a large rotational and
upward motion that leads to the interaction with the "mobile loop" of GroES.
These domain movements lead to a dramatic expansion of the inner chamber of
GroEL from 85,000 A3 to 175,000 A3 and bury the hydrophobic residues that
serve to bind the substrate. This brings about a change in the biochemical
properties of the cavity lining from hydrophobic to hydrophilic and leads to a
displacement of the substrate into the central cavity of GroEL (Xu et al, 1997). As
a result, GroELIGroES complex forms a "folding cage" that can accommodate a
protein of up to 60 kDa in size. This "folding cage" provides important structural
and biochemical features that may facilitate protein folding. First, the substrates
fold in isolation from the cytosolic environment, inside a spatially confined space,
and second, the conserved hydrophobic and negatively charged residues in the
inner lining of the GroEL folding chamber may serve as chemical chaperones to
initiate folding (Tang et al, 2006). Several cycles of substrate binding and release
by GroEL may be required for the polypeptide to reach its final folded state.
GroEL exhibits a high degree of allostery, with positive cooperation in ATP
binding within the subunits of the same ring, and negative allostery in the
opposite ring (Burston & Walter, 2005, Lin & Rye, 2006). The GroEL cycle starts
with binding of the polypeptide to the apical surfaces of the trans ring, followed by
the binding of ATP. This induces the dissociation of the polypeptide and GroES
from the opposite ring and permits the binding of GroES to the trans ring. The
trans ring now becomes the cis ring. The binding of ATP and GroES brings about
dramatic rearrangements in the cis ring and displaces the substrate into the
GroEL cavity where folding occurs. The folding proceeds until all of the ATP in
the cis ring is hydrolyzed to ADP, which primes the cis complex for disassembly
(Burston & Walter, 2005, Lin & Rye, 2006).
1 .I 5. Group II Chaperonins
The group II chaperonins are represented by CCT (also known as TriC or
c-cpn) found in eukaryotic cytosol, and thermosome, found in archaea. CCT acts
as a chaperone for a distinct subset of proteins including the cytoskeletal proteins
actin and tubulin, which have an obligatory dependence on CCT for folding
(Valpuesta et al, 2005). The archaeal thermosome is thought to have a broader
substrate specificity comparable to that of hsp70 in eubacteria (Leroux, 2001).
Unlike GroEL, CCT can bind to its substrates co-translationally, and relies on its
co-chaperonin prefoldin for binding to actin and tubulin (Spiess et al, 2004).
Like GroEL, CCT and the thermosome are doughnut-shaped structures
that consist of two stacked rings. In contrast to GroEL, which is composed of
identical subunits, both CCT and the thermosome are heterooligomers
(Valpuesta et al, 2005). Each CCT ring is composed of eight homologous
monomers that on average share a 30% sequence identity. The thermosome
42
rings, on the other hand, may be composed of one or two different subunits with
an eight-fold symmetry or three different subunits with a nine-fold symmetry
(Valpuesta et all 2005). The structure of the thermosome from an archaeon
Thermoplasma acidophilum has been solved to 2.6 A resolution (Ditzel et al,
1998). Each ring of the T.acidophilum thermosome consists of 8 alternating a-
and P-subunits which share a 60% sequence identity and superimpose with an
r.m.s.d. of 1.1 A (Figure 1.12) (Ditzel et all 1998). Each ring encloses an internal
space of approximately 130,000 A3, which is significantly smaller than the central
cavity of the cis ring of GroEL. The domain composition of the subunits in the
Group I and II chaperones is similar in that each has an apical substrate binding
domain, an equatorial ATP binding domain, and an intermediate hinge domain.
However, the monomers of the Group I1 chaperonins contain an additional a-
helical protrusion to the apical domain that plays the same role as the GroES co-
chaperonin of GroEL, namely, acting as a lid to the chaperonin folding chamber
(Figure 1.12) (Ditzel et al, 1998). ATP hydrolysis triggers the closure of the lid,
which confines the substrate inside the folding chamber (Meyer et al, 2003a).
In addition to actin and tubulin, CCT binds to several other proteins that
share no common features other that many of them contain tryptophan-aspartic
acid repeats and occur as oligomeric complexes (Spiess et al, 2004). The
substrate specificity of CCT is not well defined. Both polar and hydrophobic
sequences have been implicated in the recognition of actin by CCT (Hynes &
Willison, 2000, Rommelaere et all 1999). On the other hand, two hydrophobic
sequences that mediate binding to CCT have been identified in VHL (von Hippel-
Figure 1.12 The structure of the archaeal thermosome from Thermoplasma acidophilum.
This figure was created based on PDB entry 1A6D (Ditzel et al, 1998). A) A cartoon representation of the a-subunit of the thermosome. The P-subunit is assumes a virtually identical three-dimensional structure as the a-subunit ((Ditzel et al, 1998). The equatorial domain is in green, the intermediate domain is in red, and the apical domain is in blue. The lid-like loop on the apical domain is in yellow. B) Left pane: a surface representation of the therrnosome hexadecarner. One of the a-subunits is coloured according to the scheme in A). Other a-subunits are in violet, and P- subunits are in lilac. Right pane: a cartoon representation of the view in left pane, rotated 90" along the horizontal axis.
Lindau protein), another CCT substrate (Feldman et al, 2003). Taking into
consideration the diversified substrate specificity as well as the heterooligomeric
nature of CCT, it is possible that each CCT subunit recognizes a specific motif in
the substrate. Several studies support this hypothesis. For example, a complex
between a-actin and CCT, resolved by cryo-electron microscopy, revealed that
actin binds to the apical domains of two specific CCT subunits (Llorca et al,
1999). More recently, a study by Spiess et a1 demonstrated that the different
subunits of CCT recognize specific motifs in VHL, however, the substrate
recognition is somewhat redundant among the different subunits (Spiess et all
2006). In addition, the substrate binding site in CCT occurs at a site in the apical
domain that is structurally equivalent to that of GroEL, but because CCT is
composed of different subunits, each substrate binding site has a unique pattern
of hydrophobic and polar residues (Spiess et al, 2006). Clearly, the mechanism
of substrate recognition in CCT is different from that of GroEL, which is less
specific and generally prefers to bind hydrophobic sequences.
I .I 6. The ClplHspl00 family
The members of the ClpIhsp100 protein family belong to AAA+
superfamily of ATPases associated with various cellular activities. The proteins of
the Clplhsp100 family are found in prokaryotes and in eukaryotic mitochondria
and chloroplasts, where they are involved in protein degradation or
resolubilization of protein aggregates. To carry out this function, many Clp
chaperones, such as ClpA, ClpX, and HslU, associate coaxially with a protease,
ClpP or HslV, and form ClpAP, ClpXP, or HslUV chaperone-protease complexes
(Zolkiewski, 2006). The chaperone ClpB is different in that respect because it
does not associate with a protease and is not involved in protein degradation.
Instead, the role of ClpB is to disaggregate protein aggregates, a function which
requires cooperation with the Hsp701Hsp40 chaperone system. Unlike other Clp
chaperones, ClpB is also found in the cytosol of yeast and plants (Bosl et al,
2006).
All Clp family proteins contain one (in ClpX, HslU) or two (in ClpA, ClpB)
structurally conserved ATPase domains with Walker A and Walker B nucleotide
binding motifs. In addition, most proteins contain an extra domain, which can be
found at the N-terminus, such as in ClpA and ClpB, or inserted into the
nucleotide binding domain, such as the I-domain of HslU (Dougan et al, 2002).
These extra domains have been implicated in interaction with substrates and with
adaptor proteins, which modulate the interactions between Clp chaperones and
their substrates (Dougan et all 2002).
loop 1
Figure 1.13 The structures of the ClplHsplOO family chaperones ClpA and CIpB.
A) A cartoon representation of ClpA monomer (IKSF, (Guo et al, 2002)). The N- domain is in blue, the nucleotide binding domain (NBD) 1 is in yellow, and NBD2 is in green. B) A cartoon representation of ClpB monomer (IQVR, (Lee et al, 2003)). The colouring scheme is the same as in A), except the coiled-coil insertion is in orange.
The overall architecture and the mechanism of action are similar among
the Clp chaperones. They are oligomers and contain 6 identical subunits which
form a ring-shaped structure, with ATP-binding sites located at the interfaces
between SI- buni its. The hexameric ring contains a narrow channel in the centre,
through which Clp chaperones thread their substrates in order to force their
unfolding (Maurizi & Xia, 2004, Weibezahn et al, 2004, Zolkiewski, 2006). The
inner lining of the channel contains two constrictions, or diaphragms, formed by
six mobile loops, one from each monomer. These loops are tyrosine and glycine-
rich, conserved among the different Clp chaperones, and essential for substrate
binding and translocation. It has been suggested that the movement of the loops
might help in the translocation of the substrate through the central channel
(Hinnenvisch et al, 2005). Upon translocation, ClpA, ClpX, and HslU feed their
substrates directly into the central channel of their associated protease, whereas
ClpB releases its substrates in an unfolded form to allow their refolding, perhaps
with assistance of the Hsp70/Hsp40 chaperones (Zolkiewski, 2006).
Overall, the crystal structures of ClpA and ClpB monomers are similar:
each contains an a-helical N-terminal domain, involved in substrate and adaptor
protein binding, which is followed by the two nucleotide binding domains (NBD)
arranged in tandem (Figure 1.13AB). (Guo et al, 2002, Lee et al, 2003) In
contrast to ClpA, however, ClpB contains an 85 A long coiled-coil, mobile
insertion in its nucleotide binding domain 1, whose relative position and mobility
is essential for the protein disaggregation function of the chaperone (Figure
1.136) (Lee et al, 2003). These insertions are located on the outside of the
hexameric particle and give it a propeller-like shape; their precise function is not
clear.
ClpA and ClpX recognize and bind to the ssrA tag, an I I-residue peptide
added to the C-terminus of the proteins stalled at the ribosome (Keiler et al,
1996). ClpB, on the other hand, interacts with untagged aggregated proteins. It is
presently not clear how ClpB selects its substrates. It prefers to bind to peptides
enriched in aromatic and basic residues, with a stochiometry of one peptide per
ClpB hexamer, which points to the existence of a single centrally located
substrate binding site. A conserved residue, Tyr251, is located in the central pore
of ClpB and is crucial for substrate binding (Schlieker et all 2004). In addition,
two acidic residues found in the N-terminal domain have also been implicated in
interactions with the substrates (Barnett et al, 2005). These residues are located
adjacent to a hydrophobic groove, which may be the site of the substrate binding
in the N-terminal domain (Barnett et all 2005). An equivalent hydrophobic surface
is also found in the N-terminal domain of ClpA (Xia et all 2004). It has been
suggested that in the hexameric structure, the N-terminal domains would create
a funnel-like surface that concentrates the weakly bound substrates near the
central pore (Barnett et all 2005, Xia et al, 2004).
I .I 7. Conclusion
In this chapter, several different types of molecular chaperones were
discussed. Although not by any means exhaustive, this review of the molecular
chaperones highlights several different structural features that allow these
proteins to protect their substrates from improper interactions and ensure correct
folding or participation in a proper cellular process. There does not seem to be a
unifying scheme in terms of structural features of the chaperones, although
several broadly defined structural categories can be described. For example,
several chaperones, such as TF, Skp, SurA, prefoldin, and hsp70, can be
described as "clamps" because they tend to enclose their substrates in a binding
site via arm-like structures (reviewed in Stirling et all 2006). Others, such as the
type Ill secretion chaperones, and perhaps the Sec-dependent secretion
chaperones SecB and CsaA, are relatively rigid structures that bind substrates
via small grooves on their surfaces. The pili chaperones PapD and FimC provide
missing steric information to their substrates that may prevent the substrates'
misfolding and aggregation. Type I and II chaperonins, on the other hand,
employ a completely different mechanism of interaction with substrate, enclosing
it in a central chamber with a specific microenvironment that facilitates substrate
folding. The ClpIHsp100 family chaperones thread their substrates through a
central channel to force their unfolding.
Despite the difference of the mode of interaction with substrates, it
appears that most chaperones prefer to bind to hydrophobic sequences within
their substrates, which is consistent with the fact that exposing these residues
may lead to improper folding and aggregation of the substrate proteins. Some
chaperones, such as hsp70 and GroEL, have broad substrate specificities,
whereas others, such as type Ill secretion chaperones, are more specific.
Many chaperones contain auxiliary domains within their sequence or form
quaternary interactions with other proteins which carry out a supplementary
function. Examples are Trigger Factor and SurA, which contain a peptidyl-prolyl
isomerase domain, Hsp70, which associates with various co-chaperones that
present it with specific substrates or couple it to specific pathways, and ClpA and
ClpX, which associate with the protease CIpP, which degrades their substrates.
The structures of all molecular chaperones available from the Protein Data Bank
are summarized in Appendix B, table B1.
The molecular chaperones are of great interest because they are an
essential part of the cellular machinery and are involved in numerous cellular
processes. Many chaperones play an important role in disease, such as hsp90,
an anti-cancer drug target, or type Ill secretion chaperones, which contribute to
secretion of virulence factors in pathogenic bacteria. The structures of some
potentially important chaperones, such as BiP, an hsp70 homolog from ER, or
most of the Tat secretion chaperones, have not yet been determined. Although
some chaperones have been studied in great detail, there is still a lot to be
discovered about the way these fascinating molecular machines work.
CHAPTER 2. The Crystallographic Analysis of Bacillus Subtilis CsaA
The results of the work described in this chapter were published in
Shapova YA, Paetzel M. Crystallographic analysis of Bacillus subtilis CsaA. Acta
Crystallographica. Section D: Biological Crystallography. 2007 April; 63(Part
4):478-85.
2.1. Introduction
The Sec-dependent protein targeting and translocation pathway is
universally conserved across all three domains of life (Pohlschroder et al, 2005).
In eubacteria, secreted proteins are synthesized in the cytosol as precursors
carrying an amino-terminal signal peptide (Driessen et al, 2001). These
precursors are targeted to the translocation machinery at the cytosolic
membrane. In Escherichia coli, the translocation machinery (translocase)
involves a translocation channel composed of three integral membrane proteins
SecYEG, SecA, an ATPase that provides energy for the translocation process,
and several accessory proteins, such as SecD, SecF, YajC (Figure 2.1) (de
Keyzer et al, 2003, Driessen et al, 2001, Stephenson, 2005) and YidC (Yi &
Dal bey, 2005).
In bacteria, most of the protein secretion is carried out post-translationally
(Pohlschroder et al, 2005), with the homotetrameric SecB functioning as a
targeting factor that binds to the core regions of the newly synthesized proteins
51
co-translational secretion
post-translational secretion
) ribosome
'-- i c pre-pmtein slgnal sequence
/ 1
I cytoplasm
periplasm
Figure 2.1 A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria.
A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria. The proteins destined for secretion or insertion into the membrane are synthesized in the cytosol. and are targeted to the translocation machinery at the inner membrane. Targeting occurs co-translationally via the SRP-dependent pathway or post-translationally via the SecB and CsaA dependent pathways. SRP is a GTPase that binds to the pre-protein signal sequence as it emerges from the ribosome and targets the ribosome-nascent chain complex to the membrane. At the membrane, SRP interacts with its receptor FtsY in a GTP-dependent manner, which leads to the insertion of the pre-protein into the SecYEG channel Translation continues pushing the pre-protein across the translocation channel. SecDFNajC and YidC facilitate the assembly of proteins into the inner bacterial membrane. In the SecB- and CsaA-dependent pathways, the chaperones interact with pre-proteins post-translationally, binding to an exposed core of a pre-protein. SecB and CsaA then target their substrates to the cytosolic membrane, where they interact with the ATPase SecA. SecA mediates the insertion of the pre-protein into the SecYEG translocon and provides the energy for the translocation process. On the periplasrnic side, type I signal peptidase (SPasel) cleaves the signal peptide off the secreted proteins. The Sec-dependent secretion machinery in Gram-positive B.subtilis includes the same components described above for Gram-negative eubacteria, except it lacks a SecB homologue (Yamane et al, 2004). The diagram shown above may not represent all Gram-negative eubacteria, as some species contain only SecB or only CsaA.
and targets them to the SecA subunit of the translocase, while maintaining them
in an unfolded, translocation-competent state (Driessen et al, 2001).
Interestingly, certain species of eubacteria, and Gram-positive bacteria in
particular, lack SecB. The Gram-positive eubacterial species that has been
investigated the most from a protein secretion perspective is Bacillus subtilis
(Kunst et al, 1997).
The B.subtilis csaA gene was identified as being capable of suppressing
growth and secretion defects in E.coli secA and secB mutants (Muller et al,
1992). The B.subtilis CsaA (BsCsaA) protein restored the function of thermally
inactivated firefly luciferase in chaperone mutant strains of E.coli that lacked
functional GroEL, GroES, DnaK, and DnaJ (Muller et al, 2000a). In addition,
CsaA prevented the aggregation of thermally inactivated luciferase in vitro
(Muller et al, 2000a). Furthermore, it has been demonstrated that CsaA interacts
with the SecA subunit of the Sec translocase, as well as with a number of
secreted precursors, including B.subtilis prePhoB and pre-YvaY (Linde et al,
2003, Muller et al, 2000b). BsCsaA induced the translocation of prePhoB in E.
coli membrane vesicles containing translocation machinery from B.subtilis (Muller
et al, 2000b). More recently, it has been demonstrated that the levels of
expression of the csaA gene were upregulated 3.5 fold in response to severe
secretion stress in B.subtilis (Hyyrylainen et al, 2005). The above evidence
suggests that Bacillus subtilis CsaA acts as a secretion chaperone of the Sec-
dependent protein targeting and translocation pathway, possibly compensating
for the lack of SecB.
There are several Gram-negative species of eubacteria that have both
CsaA and SecB. One of these species is Thermus thermophilus. The crystal
structure of CsaA from T.thermophilus was solved to 2.0 A resolution (Kawaguchi
et al, 2001). The functional unit of CsaA is a homodimer, in which each monomer
is composed of a P-barrel domain which resembles an
oligonucleotide1oligosaccharide binding (OB) fold, as well as short N- and C-
terminal extensions from the P-barrel core, which form the dimer interface
(Kawaguchi et al, 2001). The OB-fold proteins are incredibly diverse and have
been shown to bind a variety of substrates, such as RNA, single stranded DNA,
oligosaccharides, and proteins (Arcus, 2002). There is no sequence or structural
similarity between CsaA and SecB, which functions as a dimer of dimers with
each monomer composed of 2 a helices and 4 P strands (Dekker et al, 2003, Xu
et al, 2000).
CsaA shares sequence and structural homology with TRBPI 11, a tRNA
binding protein, as well as with the C-terminal domain of methionyl-tRNA
synthetase (Met-RS). TRBPI I I binds to the outside corner of the L-shaped
tRNA, possibly via electrostatic interactions with the tRNA phosphate backbone
and thus may act as a structure-specific tRNA chaperone (Swairjo et al, 2000).
The C-terminal domain of Met-RS also possesses general tRNA binding ability
and serves as a dimerization domain in several species (Crepin et al, 2002).
Based on the structural similarity of these two proteins to CsaA, it has been
proposed that CsaA may possess a second, tRNA binding ability (Kawaguchi et
al, 2001).
Two crystal structures of CsaA from B.subtilis, a Gram positive
eubacterium, were solved at 2.0 A and 1.9 A resolution. These structures provide
a basis for the interpretation of previous biochemical characterization of BsCsaA,
as well as a comparison with the available TtCsaA structure from the Gram-
negative organism Thermus thermophilus. In addition, these structures may
provide more clues to the mode of binding of CsaA to its proposed substrates.
2.2. Materials and Methods
2.2.1. PCR and Cloning
Genomic DNA from Bacillus subtilis was purified using the standard
phenol-chloroform method (Sambrook et al, 1989). A region of B. subtilis
genomic DNA corresponding to the csaA gene was amplified by PCR using the
forward primer 5' g agc tga ata CAT ATG gca gtt att gat gac ttt gag aaa ttg gat
atc, incorporating the Ndel restriction site (in capital letters), and the reverse
primer 5' g aat gct cat GTC GAC tta tta tcc gat ttt tgt gcc gtt tgg gac agg ctg,
incorporating the Sall restriction site. Primers were designed using the annotated
B.subtilis CsaA sequence with the Swiss-Prot accession number P37584.
PCR reaction conditions were optimized to obtain the best yield of
products. PCR reactions were carried out in a 50 pL volume, including 1X PCR
Buffer that contained 1.5 mM MgCI2 (QIAGEN), 0.8 mM dNTP, 0.5 pmollpL each
of forward and reverse primers, 2.5 U of HotStar Taq DNA polymerase
(QIAGEN), and 0.5 pg of B.subtilis genomic DNA. PCR was carried out in a
MasterGradient thermocycler (Eppendorf) with the following steps: 95•‹C for 15
min, 94•‹C for 1 min, 62•‹C for 30 sec, and 72•‹C for 1 min. The last 3 steps were
repeated for 50 cycles, followed by incubation at 4•‹C.
The amplified fragments were cloned into the pCR2.1-TOP0 vector using
the TOPO TA Cloning Kit (Invitrogen). TOPO cloning strategy takes advantage of
the single adenosine overhang at 3'end of the product introduced by Taq
polymerase during PCR reaction. The vector supplied by lnvitrogen contains 3'-T
overhangs that enable direct ligation of Taq-amplified PCR products.
Topoisomerase I covalently bound to 3' phosphates of the TOPO vector
completes the ligation reaction and releases itself from the vector. The
recombinant plasmids were transformed into TOPIOF' chemically competent
E.coli cells. Ligations and transformations were performed using materials and
instructions provided by Invitrogen. The transformed cells were grown in Luria-
Bertani (LB) media supplemented with 100 pglmL ampicillin, and plasmids were
purified using QIAGEN Plasmid Miniprep kit.
The inserts containing B.subtilis csaA gene were excised from the TOPO
vector using restriction enzymes Ndel and Sall. The inserts were separated from
the plasmid DNA by agarose gel electrophoresis; all agarose gels were stained
with ethidium bromide and the bands were visualized under ultraviolet light at
320 nm. The bands of interest were excised from the gel and purified by QIAGEN
Gel Extraction kit. The purified inserts were ligated into the expression vector
PET-28a(+) (Novagen), predigested with Ndel and Sall. This plasmid features a
cleavable N-terminal hexahistidine tag, T7 lac promoter, multiple cloning sites,
and a kanamycin resistance marker. Ligation was carried out in a 10 pL volume
and contained 0.025 pmol of NdellSall digested PET-28a(+) vector, 0.075 pmol
of or B.subtilis csaA insert, 1.25 mM ATP, 1 U of T4 DNA ligase (Invitrogen), and
1X T4 ligase buffer (Invitrogen). The ligation reaction was incubated at 4•‹C
overnight. The recombinant plasmids (BsCsaAlpET28a) were transformed into
the E.coli Nova Blue chemically competent cells for storage and propagation.
The transformation procedure involved mixing 10 pL of the ligation reaction with
100 pL of Novablue cells, incubation on ice for 5 min, heat shock at 42•‹C for 30
sec, and incubation on ice for 2 min. Next, 250 pL of LB media was added to the
reaction tube and the tube was shaken horizontally at 37•‹C and 250 rpm for 1
hour. 250 pL of transformed cells were spread on LB agar plates supplemented
with 50 pglmL kanamycin. The plates were incubated at 37•‹C overnight. Several
colonies were screened for the presence of insert by growing the transformed
cells in LB media with 50 pglmL kanamycin, purifying the plasmid with QIAGEN
Plasmid Miniprep Kit, and digesting the plasmids with Ndel and Sall restriction
enzymes. Plasmids containing the inserts were sequenced at the UBC NAPS
Sequencing facility using the universal T7 promoter primer. The sequencing
results were identical to the annotated entry for B.subtilis CsaA. The purified
BsCsaAlpET-28a constructs were transformed into E.coli BL21(DE3) chemically
competent overexpression cells. These cells express T7 RNA polymerase and
serve as expression hosts for the genes of interest. The transformation reaction
was carried out as described for NovaBlue cells, except that the length of heat
shock was 45 seconds.
2.2.2. Overexpression and Purification
Several BsCsaNpET28aIBL21 (DE3) transformants were tested for protein
overexpression in a small-scale experiment. Several colonies were picked from
the transformation plates and grown in 3 mL of LB media containing 50 pg1mL
kanamycin for 5 hours at 37•‹C. The protein expression was induced by
supplementing the cultures with 0.5 mM IPTG and incubating the cells with
rotation at 250 rpm at 37•‹C overnight. For a negative control, the cultures were
split into two equal volumes prior to IPTG addition and IPTG was added only to
one-half of each culture. The cells were pelleted and lysed with addition of 30 pL
of 50 mM Tris pH 8.0, 100 mM NaCI, 4 pL of 10 mgImL lysozyme, and 4 pL of
1800 UImL DNase. The lysis reaction was incubated on ice for 2 hours and
analyzed by SDS-PAGE. All polyacrylamide gels for SDS-PAGE were prepared
according to the recipe described in Sambrook et al, 1989, and stained with
Coomassie Brilliant Blue. The transformants overexpressing BsCsaA protein
were stored as glycerol stocks at -80•‹C.
For large scale protein overexpression, a fresh LB agarlkanamycin plate
was streaked with BsCsaNpET28aIBL21 (DE3) and incubated at 37•‹C overnight.
The next day, an overnight culture was started from a single colony of
BsCsaNpET28a/BL21(DE3) in 50 mL of LBIkanamycin. The next day, the
overnight culture was diluted into 2 L of LBIkanamycin media and grown at 37•‹C
until the measurement of the cells' optical density at 600 nm (OD600) reached 0.6.
The culture was induced for protein overexpression with 0.5 mM IPTG at 25•‹C
for 16 hours.
The cells were pelleted by centrifugation at 4000 x g for 10 min. Harvested
cells were resuspended in 50 mL of 50 mM Tris-HCI pH 8.0, 0.3 M NaCI, 10 mM
imidazole and sonicated in a Sonic Dismembrator 500 (Fischer Scientific) with 6
bursts of 30% power for 20 seconds, with 30 sec cooling periods between bursts.
Cells were lysed in a cell homogenizer (Avestin EmulsiFlex-C3), and the cell
lysate was centrifuged at 31000 x g for 30 minutes. The supernatant containing
the overexpressed His-tagged CsaA was supplemented with 10 mM imidazole
and applied to a column containing 3 mL nickel-nitriloacetic acid beads (Ni-NTA,
QIAGEN) pre-equilibrated with 50 mM Tris-HCI pH 8.0, 0.3 M NaCI, 10 mM
imidazole. The beads were washed with 20 mL of 50 mM Tris-HCI pH 8.0, 0.3 M
NaCI, 30 mM imidazole. The bound BsCsaA protein was eluted in a stepwise
manner with 3 mL fractions of 50 mM Tris pH 8.0, 300 mM NaCI, 100-400 mM
imidazole. The resulting fractions were analyzed by SDS-PAGE. In order to
remove imidazole, fractions that contained BsCsaA were pooled together and
applied to a HiTrap Sephadex G-25 desalting column (Amersham Biosciences)
pre-equilibrated with 20 mM Tris pH8.0, 0.1 M NaCI. The concentration of protein
was measured by the bicinchoninic acid (BCA) protein assay (Pierce). The His-
tag was removed by adding 20 units of thrombin per 1 mg of CsaA and
incubating at room temperature for 16 hours. The cleaved BsCsaA was passed
over the Ni-NTA again, and the flow-through fraction was concentrated and
applied to a HiPrep 16/60 Sephacryl S-100 HR size exclusion column
(Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCI pH 8.0, 100 mM
NaCI. Fractions containing CsaA were pooled together and concentrated to 10
mg/mL using an Amicon ultra-centrifugal filter (Millipore).
2.2.3. Crystallization and Data Collection
The initial crystallization conditions were obtained by the hanging drop
vapour diffusion method using the sparse matrix screens from Hampton
Research. All drops contained I pL of protein and 1 pL of reservoir solution and
were hanging over 1 mL of reservoir solution. The initial crystallization condition
for trigonal BsCsaA crystals was condition #42 from the Hampton Research
Crystal Screen 1, which contained 50 mM Potassium dihydrogen phosphate and
20% PEG 8000. The crystallization conditions were further refined using grid
screens based on the initial hits by varying the pH, using additives, and changing
precipitant type and concentration. The optimized reservoir condition that
produced the P3*21 crystals used for the data collection was 0.1 M potassium
dihydrogen phosphate, 12% PEG 4000. The protein solution contained 12.5
mg/mL BsCsaA in 20 mM Tris-HCI pH 8.0, 100 mM NaCI. Crystals appeared
after 2 days of incubation at room temperature.
A different crystal condition was discovered when screening BsCsaA in a
high salt buffer against the sparse matrix screens from Hampton Research. The
reservoir condition that produced the crystals in space group P4212 was
condition #30 from Crystal Screen 1, which contained 0.2 M ammonium sulfate
and 30% PEG8000. The protein solution contained 9 mg/mL BsCsaA in 20 mM
Tris pH 8.0, 1 M NaCI. The crystals appeared after 7 days of incubation at 18•‹C.
Prior to data collection, the crystals were transferred into a cryoprotectant
solution that contained the mother liquor in which 25% of water was replaced
with glycerol. The diffraction data were collected at the Simon Fraser University
Macromolecular X-ray Diffraction Data Collection Facility using a MicroMax-007
rotating anode microfocus generator operating at 40 mV and 20 mA, VariMax Cu
HF optics, X-stream 2000 cryosystem, and R-AXIS IV++ imaging plate area
detector (MSC-Rigaku). All data were collected and processed using the
Crystalclear software pack (Pflugrath, 1999). The trigonal crystals (P3*21)
diffracted to beyond 2.0 A resolution. The tetragonal crystals (P42,2) diffracted to
beyond 1.9 A resolution. Complete data sets were collected for each crystal form.
See table 1 for the data collection statistics.
2.2.4. Structure Determination and Refinement
The structures of B.subtilis CsaA were solved by molecular replacement
using the program Phaser (McCoy et all 2005) of the CCP4 suite of programs.
The coordinates of Thermus thermophilus CsaA, chain A (1GD7A) were used as
a search model. Several rounds of restrained refinement with REFMAC5
(Murshudov et al, 1997) and manual adjustment and manipulation using Coot
(Emsley & Cowtan, 2004) were used to build the BsCsaA models. CNS (Brunger
et al, 1998) was utilized as an additional tool to carry out the combined simulated
annealing, energy minimization, and B-factor refinement. The final models were
obtained by running restrained refinement in REFMACS with TLS restraints
obtained from the TLS motion determination server (Painter & Merritt, 2006). The
quality of the final models was assessed with the program PROCHECK (Morris
et al, 1992). The coordinates for BsCsaA in space groups P3221 and P4212 were
deposited into the Protein Data Bank (Berman et all 2000) with accession
numbers 2NZ0 and 2NZH, respectively.
2.2.5. Structural Analysis
Superimpositions were carried out using the program Superpose (Maiti et
al, 2004). The surfacelbinding pocket analysis was carried out by CASTp
(Binkowski et all 2003) using a 1.4 A probe radius. The mapping of the sequence
conservation onto the three-dimensional structure was performed with
CONSURF (Glaser et al, 2003). The figures were made using PyMol (DeLano,
2002). The sequence alignment analysis (Figure 2.1 0) was prepared by ClustalW
(Thompson et al, 1994) and ESPript 2.2 (Gouet et al, 2003). The protein-protein
interaction server was used to analyze the dimer interface (Jones & Thornton,
1995). The surface electrostatic analysis was performed using the vacuum
electrostatics utility in the program PyMol.
2.3. Results and Discussion
2.3.1. PCR and Cloning
[MgC121
annealing T
DNA, CIS
Figure 2.2 The optimization of the PCR amplification of B.subtilis csaA gene.
PCR reaction was carried out in several conditions with varying temperatures of the annealing step and MgCI, and DNA concentrations. The bands corresponding to BsCsaA PCR products are identified with an arrow. 1% agarose gel was used to separate the PCR products.
The B.subtilis genomic DNA sequence corresponding to the csaA gene
was successfully amplified by PCR, producing bands which correspond to the
size of the annotated csaA gene. The results of PCR optimization are illustrated
in Figure 2.2. The PCR condition that produces the most amount of product with
the least amount of non-specific amplification contained 0.5 l ~ g of B.subtilis
genomic DNA and 1.5 mM MgCI2, with the annealing step temperature of 62•‹C.
Figure 2.3 The results of cloning the PCR-amplified B.subtilis csaA gene into pCR2.1- TOP0 vector.
The plasmids from 6 colonies of E.coli TOPIOF' cells transformed with BsCsaA- Topo construct were purified, digested with restriction enzymes Ndel and Sall to liberate the insert, and separated on 1% agarose gel. Lane 1 contains 1 b.p. DNA ladder, lanes 2-7 contain the digested constructs, and lane 8 contains the mass ruler.
Cloning of PCR-amplified csaA gene into the pCR2.1-TOP0 vector was
successful as the restriction enzyme digest of the constructs liberated bands
similar in size to the csaA gene (333 b.p.) (Figure 2.3).
Figure 2.4 The results of subcloning of the csaA gene fragment into the expression vector pET28-a(+).
The plasmids from 6 colonies of E.coli NovaBlue cells transformed with BsCsaA- pET28a construct were purified, digested with restriction enzymes Ndel and Sall to liberate the insert, and separated on 1% agarose gel. Lane 1 contains 1 b.p. DNA ladder, and lanes 2-7 contain the digested constructs.
The subcloning of csaA fragments excised from BsCsaA-Topo construct
and ligated into the overexpression plasmid pET28-a(+) was successful as the
restriction enzyme digest of the constructs liberated bands similar in size to the
csaA gene (333 b.p.) (Figure 2.4).
The sequencing of the resulting BsCsaA-pET28a construct revealed a
100% match to the annotated sequence of B.subtilis csaA gene, as well as a
partial sequence corresponding to the pET28-a(+) vector.
2.3.2. Overexpression and Purification of BsCsaA
Figure 2.5 A small-scale induction of BsCsaA expression from E.coli BL21(DE3) cells transformed with the BsCsaA-pET28a construct.
In lanes indicated by (+), cells were induced for protein expression with 0.5 mM IPTG. In lanes indicated by (-), cells were treated similarly, except no IPTG was added. Cells were lysed in a lysis buffer that contained lysozyme, and the lysates were separated on a 15% SDS-PAGE and stained with Coomasssie Blue.
The small-scale induction was successful, as the cells induced with IPTG
showed a very large band on the SDS-PAGE, whereas the cells that were not
induced did not show such band (Figure 2.5). The size of the overexpressed
protein is roughly 14 kDa, similar to that of the His-tagged BsCsaA protein
s u ~ t ft wash Elutions imidazolt
std p 10 10 30 '100 200 300 400 5 0 0 ' ~ ~
20 kDa
6 kDa
Figure 2.6 Purification of BsCsaA protein by nickel affinity chromatography.
The fractions obtained by nickel affinity chromatography were analyzed for protein content on 15% SDS-PAGE (stained with Coomassie Blue). Std: broad range standard; p: pellet obtained by centrifugation of lysed cells; supt: supernatant obtained after centrifugation of lysed cells; ft: flowthrough from the Ni-NTA column. The bands corresponding to BsCsaA protein are indicated with an arrow.
After centrifuging the cell lysates, the majority of the overexpressed
BsCsaA protein remained in the soluble form (Figure 2.6). His-tagged BsCsaA
efficiently bound to Ni-NTA beads, whereas most of the contaminated proteins
eluted in the flowthrough. BsCsaA was efficiently removed from the column with
addition of increasing concentrations of imidazole. Most of BsCsaA eluted when
buffer containing 200-400 mM imidazole was added. The eluted BsCsaA protein
was sufficiently pure, with very few of contaminating proteins present (Figure
2.6).
thrombinl
MGSSHHHHHHSSGLVPRGSHMAVIDD 6His tag
Figure 2.7 Optimization of thrombin digest of BsCsaA.
BsCsaA was incubated with 5-20 units of thrombin at 4" and at room temperature overnight. The digestion products were separated on 20% SDS-PAGE (stained with Coomassie Blue). The sequence above the gel indicates thrombin cleavage site within the N-terminal hexahistidine tag. The residues in bold indicate the N-terminus of the annotated B.subtilis CsaA sequence.
RT, OIN a, a c F e
In order to improve the crystallization efforts, the N-terminal hexahistidine
(His) tag of BsCsaA was cleaved off with thrombin. Thrombin cuts specifically at
a site within the His tag indicated by an arrow (Figure 2.7). The conditions that
produced a complete cleavage of the His tag involved digestion at room
temperature overnight using 20 U of thrombin per mg or BsCsaA (Figure 2.7).
: P % z 0 5 10 15 20 5 10 15 20 thrombin, U
- e mE w o C U
4', OIN
Figure 2.8 Purification of BsCsaA by size exclusion chromatography.
A) A profile of BsCsaA elution from the HiPrep 16/60 Sephacryl S-100 HR size exclusion column (Amersham Biosciences). The flow rate was 1 mL/min; 4 mL fractions were collected. B) The fractions corresponding to BsCsaA peak were collected, concentrated, and analyzed on a 20% SDS-PAGE stained with Coomassie Blue.
The digested BsCsaA protein was further purified by size exclusion
chromatography. BsCsaA eluted from the size exclusion column as a single
peak, indicating that no substantial amounts of contaminating proteins were
present (Figure 2.8). Fractions 37-41 were collected, combined, concentrated,
and used in subsequent crystallization experiments.
Crystallization and Data Collection
P 322 I P3221 0.05 M ammonium formate, 0.1 M potassium dihydrogen 20% PEG 8000 phosphate, 12% PEG 4000
P42,2 0.1 M ammonium sulfate, 30% PEG 8000
Figure 2.9 Crystals of B.subtilis CsaA
Space groups and reservoir conditions are indicated below the picture of each crystal.
Several different reservoir conditions produced crystals of sufficient size
and quality for X-ray data collection experiments (Figure 2.9). BsCsaA was
successfully crystallized in two space groups: P3221 and P4212. The fact that the
crystals formed in two different space groups is most likely due to different salt
concentrations in buffers that the proteins were kept in prior to setting up the
crystal drops. For crystallization in space group P3221, the protein was kept in 20
mM Tris pH 8.0, 0.1 M NaCI. For crystallization in space group P42,2, a high salt
buffer was used (20 mM Tris pH 8.0, 1 M NaCI).
The crystals shown in middle and right panes of Figure 2.9 were used for
X-ray data collection and subsequent structure determination. Trigonal crystals
(P3221) diffracted to beyond 2.0 A resolution; tetragonal crystals (P4212)
diffracted to beyond 1.9 A resolution. The data collection statistics are
summarized in Table 2.1.
Table 2.1 The crystallographic data collection statistics for B.subtilis CsaA.
DATA COLLECTION Space group P322 1 P 4 2 , 2 Unit cell dimensions (A) 148.4 x 148.4 x 54.1 109.2 x 109.2 x 37.4 Resolution range (A) 28.05 - 2.00 (2.07 - 54.57 - 1.90 (1.97 -
2.00) 1.90) Total number of reflections 209396 190906 Number of unique reflections 45675 17190 Average redundancy 4.58 (4.25) 1 1.1 1 (9.99) Oh completeness
# 98.6 (97.0) 93.3 (87.0)
Rrnerge 0.044 (0.310) 0.051 (0.286) Ilol 20.4 (4.6) 31.9 (7.2)
Values in parentheses are for the highest resolution shell. #
R m s r g e = XI1 - (I)I/X(I), where I is the observed intensity obtained from multiple observations of
symmetry-related reflections after rejections.
2.3.4. Structure Determination and Refinement
The collected diffraction data were used to solve the structures of BsCsaA
proteins. Molecular replacement using T.thermophilus CsaA structure as a
search model was utilized to solve the structures of BsCsaA. For the P3221
structure, the solution was obtained with 4 molecules in the asymmetric unit, and
for the P4212 structure, the solution was found with 2 molecules in the
asymmetric unit. The solvent content in the trigonal crystal was 65.7%, and in the
tetragonal crystal, 47.0%. Several cycles of manual adjustment with Coot and
refinement with REFMACS produced good quality models with sufficiently low R
values of 0.192 and 0.202, for the P3221 and P42,2 structures, respectively. Low
R values indicate good agreement with experimental data. Solvent atoms (water
and glycerol) were modeled in to improve agreement between the model and the
experimental results. Several residues could not be modeled due to a lack of
electron density, indicating a high degree of thermal motion. These residues
occurred at the extreme N-terminus of CsaA, and in loop 24-28. Those are most
likely flexible structures that do not assume a fixed position in the crystal. Root
mean square deviations of bonds and angles were sufficiently close to ideal
values. The Ramachandran plot analysis was utilized to assess the main-chain
conformational angles of the proteins. The main-chain angles of a vast majority
of non-glycine residues are sterically favoured, and no non-glycine residues
occur in the sterically disallowed area of the Ramachandran plot (Appendix B,
Figures B2 and B3). The summary of refinement and structure validation
statistics can be found in Table 2.2.
Table2.2 A summary of refinement statistics for the models of B.subtilis CsaA structure.
REFINEMENT P322 1 P42,2
Molecules in asymmetric unit 4 2
Number of protein atoms 3306 1654
Number of solvent atoms 320 123
Water 284 116
Glycerol 36 7
Rwork 0.192 0.202
b e e 0.230 0.245
r.m.s.d. bond lengths (A) 0.019 0.017
r.m.s.d. bond angles (") 1.78 1.61
Average B-factor (A2) - protein 17.4 17.2
Average B-factor (A2) - solvent 32.0 32.5
RAMACHANDRAN ANALYSIS (%)
Favoured 91.5 90.3
Allowed 8.5 9.1
Generous 0.0 0.6
Disallowed 0.0 0.0
Residues missing from the models Chain C: 24-28 Chain B: 1-2
due to a lack of electron density
Residues modeled as alanines due to Chain C: 32,40,91 Chain A: 1
a lack of side-chain density Chain D: 8, 22,40 Chain B: 25,27
+ R, = ZIIF~ -IF~II/ZIF~ SRf,, is calculated the same way as R factor for data omitted from refinement (5% of reflections for all data sets).
2.3.5. Sequence Alignment Analysis
h 1 s l s? h2 a3 hS B . subtilis-CsaA U - -R.QQR- eana +
W I V #AM M U HAT Iur ULT HAT 3'1s Ef S I t + LCD rzs Mt? TIE X I % RTP LID SDP YVR LYD LiC t V A i
4 a 5 sh %7 \ X + I - - - + + +
Legend t E - 3D slruchrm avrulabb - Gram-parrtlive eubactada N - Gram-nqptive eubadaria
-Archoe0 4 - inhfchain hydrogen brding through ba8bone ato& A - interchain hydrogen bonding throush rida chain at- * - backbone aiomr prrtidpate in binding site farmalion * -side chain atom patiidpate in binding site formation a . similar residues 1 -Hal rsaklrsaklm
Figure 2.10 Sequence alignment of BsCsaA and other CsaA proteins and the tRNA binding proteins MetRS and TRBPI 11.
The percent identity of each protein with BsCsaA is listed at the bottom right of each sequence. The secondary structure of BsCsaA as determined by DSSP (Kabsch & Sander, 1983) is above the alignment. The sequence numbering is that of BsCsaA. The figure below the alignment represents a stereo view of Ca trace of the BsCsaA monomer. The location of every 10th residue is indicated by a corresponding number. Portions of the polypeptide that participate in the binding site formation are shown in red.
There are three residues that appear universally conserved among the
CsaA, TRBPI 11, and MetRS proteins: Gly38, Asn69, and Ser80 (Figure 2.1 0).
Asn69 and Ser80 have been identified as residues crucial for tRNA binding in
TRBPI 11 (Swairjo et all 2000). The following residues appear to be conserved in
most CsaA proteins and are different in TRBPI 11 and MetRS: 26, 29-30, 42, 46-
51, 70, 83, 86. Based on the phylogenetic tree analysis of 18 CsaA and 18
TRBPI 11 and MetRS (C-terminal domain only) proteins (Appendix B, Figure BI),
CsaA proteins are distinct from the other group and form their own subfamily.
It is notable that the csaA gene is found in many species of Gram-positive
and Gram-negative eubacteria, as well as archaea. In the Gram-positive
eubacteria, csaA seems to be present only in the species of the genus Bacilli and
Clostridia.
2.3.6. Structural Overview
CsaA from B. subtilis (BsCsaA) is a homodimeric molecule, in which each
monomer is 110 amino acids long and has a molecular weight of 12 kDa. The
core structure of each monomer displays a well described oligonucleotide I
oligosaccharide binding (OB) fold, a 5-stranded P-barrel with a short capping a-
helix (Murzin, 1993). In the case of BsCsaA, the P-barrel is formed by strands s l ,
s2, s3, s4, s7, and an a-helix h3 located between s3 and s4 (Figure 2.1 IA). An
additional short helix h2 is found in the loop region between s2 and s3. Two short
P-strands, s5 and s6, hydrogen bond to each other and are connected by a type
II p-turn. In addition to the P-barrel, an a-helix h l is found at the N-terminus of the
protein, and the C-terminus contains strands s8 and s9. These elements
res.
Figure 2.1 1 The structure of BsCsaA.
A) A cartoon diagram of BsCsaA. Chains A and B are shown in purple and orange, respectively. (B) A Ca trace of the six superimposed chains from the two structures of BsCsaA, coloured by B-factor. Areas with the lowest B-factor are colored blue, and areas with the highest B-factor are colored red. (C) A Ca trace of the superimposed dimeric structures of BsCsaA (blue) and TtCsaA (red). Regions that show different conformations in different chains are labelled.
I participate in the formation of the dimeric structure of CsaA. I The two structures of BsCsaA differ somewhat, particularly in the positions
of atoms in residues 23-32 and 73-79 (Figure 2.116). These regions contain
residues that contribute to the formation of the putative substrate binding site.
The four chains in the asymmetric unit of the structure from the trigonal crystals
(P3221) superimpose over the backbone atoms with a root mean square
deviation (r.m.s.d.) of 0.3 A. The two chains in the asymmetric unit of the/
structure with the spacegroup P4212 superimpose with r.m.s.d. of 0.7 A. The
r.m.s.d. of superposition of all 6 chains over backbone atoms is 0.4 A.
The structural neighbours of B. subtilis CsaA were found by performing a
VAST search of the medium redundancy PDB database (Gibrat et al, 1996). The
closest structural neighbour is the CsaA protein from T thermophilus, with a
r.m.s.d. of superposition over the backbone atoms of 1.6 A (Figure 2C). Other
structural neighbours include the tRNA-binding protein TRBPI 1 1, the C-terminal
domain of methionyl-tRNA synthetase, a MetRS related protein, and the EMAPll
domain of the p43 protein from the human aminoacyl-tRNA synthetase complex. / The r.m.s.d. of superposition with these proteins and BsCsaA ranges from 2.1 A
to 3.8 A, while the sequence identity ranges from 27% to 48% (Table 2.3) - I
Notably, all of the structures described above contain tRNA binding domains.
Table 2.3 The structural neighbors of B.subtilis CsaA
1 1 PXF
Protein
CsaA
MetRS, C-
terminal
domain
TRBPI I I
EMAPll RNA
binding
domain of the
P43 Protein
TRBPI I I
Residue Rmsd*, Seq ID•˜, Organism
Range A YO
Thermus 2-109 1.57 48
thermophilus
Pyrococcus 7-112
horikoshii
Aquifex I aeolicus I
Homo 1 sapiens
Escherichia
coli 1 4-110 1 3.79
27 1 *Root mean square deviation of superposition to BsCsaA structure. 'percent sequence identity to BsCsaA.
2.3.7. The Dimerization Interface
The two monomers of BsCsaA are held together by 19 hydrogen bonds
(Table 2.4). Since the dimer has a local two-fold axis of symmetry, the same
residues form the hydrogen bonding interactions in both chains. The majority of
the inter-chain hydrogen bonding network is localized to the C-terminal portion of
the protein, and occurs through mainchain atoms (Figure 2.12C). The hydrogen
bonds that participate in dimerization are listed in Table 2.4. On average, 1550
a* (about 22.5%) of total accessible surface area is buried in the interface, which
is comprised of mostly non-polar atoms.
Figure 2.12 Dimerization of BsCsaA via hydrogen bonding interactions.
A) A fragment of the 2F,-F, electron density at 1.00, demonstrating dimerization interactions via Tyr54 hydrogen bonding to Asp100'. B) A fragment of the 2F,-F, electron density at 1.00, demonstrating dimerization interactions in the P3,21 structure via Ala2 hydrogen bonding to Asn69'. C) A ribbon diagram of BsCsaA dimer, showing the location of residues participating in dimer formation via hydrogen bonding. Residues participating in main-chain hydrogen bonds are indicated in blue. Residues participating in side-chain hydrogen bonds are shown as green sticks, and the hydrogen bonds that they form are in red.
There are two notable hydrogen bonding interactions that occur in
residues near the N-terminus. There is a hydrogen bond between the side chains
of Lys9 and Asp6' (and Lys9' and Asp6, respectively). In the P4Z12 structure, this
hydrogen bond is formed directly, from the NZ of Lys9 to the OD1 of Asp6.
However, in the P3221 structure, this hydrogen bond is indirect and occurs
through a water molecule (W151). All 4 residues (Lys9, AspG', Lys9', and Asp6)
make hydrogen bonds to water W151. The structure P3221 has two hydrogen
bonds that occur at the N-terminus, between the backbone atoms of Ala2 and
Asn69' (and vice versa) (Figure 2.12B). This interaction is not observed in the
P4Z12 structure.
Among the residues that make hydrogen bonding interactions through
their side chains, Tyr54 is notable because this residue is highly conserved
among the CsaA proteins, TRBPI 11, and the C-terminal portion of MetRS, all of
which are dimers. Tyr54 forms hydrogen bonding interactions with AsplOO
(Figure 2.12A). Although AsplOO is conserved to a lesser degree than Tyr54,
most proteins contain residues at this position capable of providing a hydrogen
bond acceptor for Tyr54. It is therefore possible that Tyr54 may be important for
the CsaA dimerization.
Table 2.4 The interchain hydrogen bonds between the two monomers of BsCsaA.
Donors Acceptors
Residue Atom Residue Atom
'Ala 2 N Asn 69' 0
#LYS 9 NZ Asp 6' OD1
Arg 53 NH1 Gln 101' OEl
Tyr 54 OH Asp 100' OD2
Lys 62 NZ Asp 100' 0 D2
Gly 86 N Gly 110' 0
Ile 88 N Lys 108' 0
Gln 98 N Gln 98' 0
• ˜ ~ l n 98 NE2 Gln 98' OEl
Asp 100 N Leu 96' 0
Gly 110 N Gly 86' 0
The inter-chain hydrogen bonds between the two monomers of BsCsaA were obtained from the optimal hydrogen bonding network (Hooft et al, 1996). These hydrogen bonds occur in both P3221 and P42,2 crystal forms. Due to a local two-fold axis of symmetry in the dimer, the same residues form hydrogen bonds in both chains of the dimer (except as noted). 'occurs only in P3,21 structure # occurs only in P42,2 structure ' occurs only once in each dimer
2.3.8. The Potential Substrate Binding Site
The CastP (Binkowski et all 2003)) analysis of the solvent accessible
surface revealed that BsCsaA contains two large T-shaped cavities, one in each
monomer. The two binding sites are separated by a rotation of approximately 90
degrees about the axis of the dimer (Figure 2.13A). These cavities are located on
one side of each P-barrel, and are formed predominantly by loops. The following
residues contribute to the binding cavity formation: 26-30, 46-52, 70-73, 75, 80-
84, 86, 88, 92, 94, 96, 110'. The cavity dimensions are approximately 15 x 15 A,
with a depth ranging from -3 to 6 A. The residues forming the cavity are mostly
Figure 2.13 The potential substrate binding sites in BsCsaA.
(A) A cartoon representation of the BsCsaA dirner with the substrate binding cavities shown as surface. Carbons are shown in green, oxygens in red, and nitrogens in blue. B) The superimposition of the residues forming the binding site from the six chains of the two structures of BsCsaA. The surface corresponds to the putative substrate binding cavity of BsCsaA structure in space group P42,2, chain A.
hydrophobic and come from the same monomer, except for the C-terminal
GlyllO. The residues that line the floor of the cavity are Ser46, Ser47, Ala48,
lle50, Ser80, Glu81, Va182, Va184, and Leu96. The three serines (Ser 46, 47, and
80) form a hydrophilic patch in the center of the cavity floor. The cavity walls are
formed by Ala26, Va128, Gln49, Phe70, Pro71, Pro72, Arg73, lle75, Leu83,
Gly86, lle88, Va194, and Glyl 10'. Most of the residues forming the binding site do
not show large variation in the position of their atoms among the six chains seen
in the two BsCsaA structures, however, the following residues show large
variation in position: Ala26, Va128, Pro29, Phe70, Pro71, Pro72, Arg73, and He75
(Figure 2.13B). These residues are located predominantly in loops that together
82
form one wall of the binding cavity. These regions demonstrated weaker electron
density and higher B-factors, which is consistent with greater mobility in this
region. These residues superimpose with a r.m.s.d. of 1.4 A (over all atoms),
whereas the r.m.s.d. for all residues of the binding site is 0.9 A, and that for all
atoms in the six models is 0.8 A. It has been previously demonstrated that CsaA
has an affinity for binding multiple peptides (Linde et al, 2003). It is possible that
the flexibility of this wall is important to accommodate the binding of a variety of
peptide substrates, which is consistent with the general chaperone function.
It has been previously shown that BsCsaA has higher affinity to denatured
peptides, thus indicating preferred binding to unfolded proteins (Linde et al,
2003). The hydrophobic nature of the binding cavity is consistent with the
chaperone activity of BsCsaA, allowing it to bind the exposed hydrophobic
residues in an unfolded protein substrate. The hydrophilic patch in the floor of the
binding cavity, formed by the three serines (Ser 46, 47, and 80), provides the
possibility for the hydrogen bonding interactions between the residues of the
cavity and the backbone atoms of the protein substrate in an extended
conformation. It is possible that the protein substrate wraps around the surface of
CsaA, much like the substrate-chaperone interactions recently described for
SecB (Crane et all 2006).
A docking experiment using the Gramm-X web server (Tovchigrechko &
Vakser, 2006) was carried out to further explore this hypothesis. Because there
is no structure of BsCsaA substrate available, a structure of the chaperone-
binding domain of YopE from a complex with a type Ill secretion chaperone
Figure 2.14 Docking of BsCsaA structure with a peptide in extended conformation.
Docking was carried out using the Gramm-X web server (Tovchigrechko & Vakser, 2006). The dimeric structure of BsCsaA in space group P3*2l, chains A and 6, was used as a receptor structure. The structure of the 55 residue long chaperone binding domain of YopE (PDB ID 1 L2W (Birtalan et al, 2002)) was used as a ligand. Prior to docking, all residues in the YopE structure (except prolines and glycines) were converted to alanines to optimize docking. The resulting docking model is shown in the figure above. BsCsaA is shown as surface, with carbons in green, oxygens in red, and nitrogens in blue. YopE is shown as sticks, with carbons in pink, oxygens in red, and nitrogens in blue. The locations of the putative substrate binding sites are identified by arrows.
SycE (PDB ID 1L2W, reviewed in section 1.9) was used as a model substrate.
This 55 residue long polypeptide is well suited for docking to BsCsaA because
most of it wraps around its chaperone SycE in an extended conformation. In
addition, the SycE chaperone is similar in size to BsCsaA. The docking model
revealed that the substrate interacts with BsCsaA in the close vicinity of both
putative substrate binding sites and wraps around the opposite side of the
chaperone in going from one substrate binding site to the other (Figure 2.14).
Due to limitations of the rigid body docking method, the conformational flexibility
of the ligand in not taken into account during docking, and therefore the substrate
is not seen to enter the potential substrate binding sites of BsCsaA in this model.
However, the model confirms that it is possible for the substrate to wrap around
BsCsaA using small grooves on its surface. In the in vitro or the in vivo situation,
it is very possible that the substrates enter both putative binding sites on BsCsaA
and may take several possible paths to wrap around the surface of the
chaperone, just as was described for SecB ((Crane et al, 2006).
Overall, the residues of the binding site are well conserved among the
sequences of CsaA proteins (Figure 2.1 0). However, some residues that are well
conserved among the CsaA proteins are different in TRBP111 and MetRS, such
as Pro29, Ser46, Gln49, Thr51. Swarjo et al identified TRBPl l I residues
important for tRNA binding (Swairjo et al, 2000). It's worth noting that most of
these residues are conserved among TRBP and CsaA, such as Ser80 (Ser82 in
TRBPIII), Arg73, and Asn69. However, two of these residues are not
conserved in CsaA proteins: Met82 (Leu83 in BsCsaA) and Glu45 (He41 in
BsCsaA). Mutating these residues in E. coli TRBP l l l reduced the binding
affinity of ~ R N A ~ ~ ~ 8-fold and 66-fold, respectively (Swairjo et al, 2000). It has
been proposed that CsaA may bind dual substrates: pre-proteins and tRNA
(Kawaguchi et all 2001). While it is possible that CsaA is capable of binding
tRNA due to its structure and sequence similarities to other tRNA-binding
proteins, the tRNA-binding ability of CsaA has not yet been demonstrated.
2.3.9. The Electrostatics and Conservation Analysis
The electrostatics analysis of the protein surface revealed two prominent
regions of electrostatic surface potential in the vicinity of the binding cavity
(Figure 2.15BC). These two areas of positive and negative electrostatic surface
potential flank the opposite sides of the binding cavity. The negative surface
potential occurs near the entrance to the binding cavity and is formed by Asp5,
Asp6, Glu8, Aspl l , and the C-terminal carboxylate (GlyllO). This negative
surface potential is consistent with proposed preference of CsaA to bind
positively charged peptides (Linde et al, 2003). An area of positive electrostatic
surface potential arises due to a cluster of basic residues at the ridge
surrounding the binding site: Arg27, Arg73, Arg74, Lys32, Lys44, and Lys79.
The electrostatics in the vicinity of the binding site differs somewhat in
BsCsaA and TtCsaA (Figure 2.15BC). The area of negative electrostatic potential
is weaker in TtCsaA than in BsCsaA and its location is shifted. This is due to the
replacement of Asp6 and Glu8 with Ala and Gln, respectively, in TtCsaA.
BsCsaA, on the other hand, contains a lysine at position 52 and a glycine at
position 90 instead of glutamic acids in TtCsaA. These replacements are
responsible for different positions of negative surface potentials in the vicinity of
the binding sites of BsCsaA and TtCsaA.
The analysis of the BsCsaA surface coloured by the conservation score
(Figure 2.15A) reveals that the following residues are highly variable: lle4', ASPS',
Glu8', Lys52, lle88, Gly90, Gln91, Asp93, GlyllO'. It is interesting to note that
these variable residues occur in a region that overlaps the area of the negative
T thermophilus
Figure 2.15 The conservation and surface electrostatic properties of BsCsaA.
A) The surface representation of BsCsaA, coloured by the conservation score, with the most conserved residues coloured dark blue, and the least conserved residues coloured red. The figure was made using ConSurf (Glaser et al, 2003). The conservation scores were obtained from sequence alignment of 36 CsaA, TRBP, and MetRS (C-terminal domains only) sequences. B and C) The protein surface electrostatics of T.thermophilus CsaA (0 ) and B.subtilis CsaA (C). Areas colored in white, red, and blue correspond to neutral, negative, and positive surface electrostatic potentials, respectively.
electrostatic surface potential at the entrance to the binding site in BsCsaA. This
region has a different pattern of electrostatic potential in TtCsaA. Based on the
high sequence variability of this region, it is possible that each CsaA protein has
its own unique pattern of electrostatic surface potential in the vicinity of the
binding site entrance, which might be optimized for the interactions with their
respective substrates.
2.4. Conclusion
CsaA is a small, dimeric protein that is present in some species of Gram-
negative and Gram-positive eubacteria and archaea. The available biochemical
data indicates that CsaA may act as a chaperone in the Sec-dependent protein
secretion system. The structure of CsaA from the Gram-positive eubacterium
B.subtilis is similar to that previously solved in the Gram-negative eubacterium T
.thermophilus. The dimeric structure is held together by 19 hydrogen bonds that
are mostly localized to the C-terminus. Seventeen of the hydrogen bonds are the
same in both P3221 and P42,2 structures, 2 hydrogen bonds are unique to each
structure. Analysis of the proposed substrate binding site reveals that it is mostly
hydrophobic with several residues forming a hydrophilic patch, which may allow
binding of unfolded peptides in an extended conformation. One wall of the
proposed binding cavity appears to be flexible, which may allow CsaA to bind a
broad spectrum of unfolded pre-protein substrates. The presence of an area of
negative surface potential near the entrance to the binding site is correlated with
the preference of CsaA to bind positively charged peptides. A region of negative
electrostatic surface potential at the entrance to the binding site in BsCsaA
contains residues that are highly variable among the sequences of CsaA,
TRBPI 11, and C-terminal regions of MetRS.
CHAPTER 3. CLONING, OVEREXPRESSION, PURIFICATION, CRYSTALLIZATION, AND REFINEMENT OF THE CRYSTAL STRUCTURES OF AGROBA CTERIUM TUMEFA CIENS CSAA
The work described in this chapter was performed in part by Dr. Anat
Feldman. My contribution to this body of work includes cloning the A.tumefaciens
CsaA construct without a peptide (AtCsaA), overexpression and purification of
the AtCsaA protein, obtaining initial crystals, as well as final refinement of the
structures of Ahmefaciens CsaA with and without a peptide.
3.1. Introduction
The components of the Sec-dependent secretion system (reviewed in
section 2.1) are similar in Gram-positive and Gram-negative eubacteria, except
for the fact that Gram-positive eubacteria lack SecB, a Sec-dependent secretion
chaperone (Yamane et al, 2004). Instead, many Gram-positive eubacteria, such
as B.subtilis, contain CsaA, another chaperone with export-related activities
(discussed in section 2.1). CsaA occurs in several Gram-positive and Gram-
negative eubacterial species, as well as in some archaea (discussed in section
2.3.5).
Agrobacterium tumefaciens is a Gram-negative eubacterium and an
important plant pathogen whose Ti plasmid is used as a valuable tool in plant
genetic engineering (Prescott et al, 2002). Unlike B.subtilis, which lacks the Sec-
dependent secretion chaperone SecB, or E.coli, which lacks CsaA,
Agrobacterium tumefaciens is one of several species that harbour both these
chaperones. CsaA protein from A.tumefaciens has 64% sequence identity to
CsaA from B.subtilis, and 48% sequence identity to CsaA from T.thermophilus.
Two crystal structures of A.tumefaciens CsaA were solved to 1.55 A and
1.65 A resolution (Feldman A. et a/, to be published). The structure (X15-AtCsaA)
that was refined to 1.65 A resolution, features a 15-residue long peptide
occupying a deep hydrophobic pocket on the surface of CsaA. The peptide was
selected from a linear peptide library displayed at the amino-terminus of phage
coat protein pVlll and showed significant binding to CsaA as determined by
ELISA. The 15-residue sequence corresponding to the selected peptide was
genetically fused to the N-terminus of CsaA prior to crystallization, using a
rationale that the N-terminus was located in close vicinity of the proposed
substrate binding site. The peptide I pocket interactions seen in the crystal
structure of XIS-AtCsaA might imitate the interactions between CsaA and its
natural pre-protein substrates. The other structure (AtCsaA), that was refined to
1.55 A resolution, was obtained from an AtCsaA construct without a peptide
tethered at the N-terminus. The two structures with and without the peptide are
compared and their pockets are analysed in detail. In addition, crystal structures
of A-tumefaciens CsaA are compared to the previously available structures of
CsaA from T.thermophilus and B.subtilis.
3.2. Materials and Methods
3.2.1. PCR and Cloning of AtCsaA and X I 5-AtCsaA
A sequence corresponding to csaA gene was amplified by PCR from
A.tumefaciens genomic DNA. The primers for PCR were designed based on a
Swiss-Prot annotated sequence with accession number Q8UDB9. PCR was
carried out with the forward primer 5' CAT ATG agc ggc gaa att tcc tat gcc gat
ttc, incorporating Ndel restriction site (in capital letters), and the reverse primer 5'
AAG CTT tca gca cat ctt ctc acc gtt cgg cac agg, incorporating Hindlll restriction
site. PCR conditions were optimized to obtain an optimal yield of products. Each
reaction was carried out in a 50 pL volume, including 1X PCR Buffer containing
1.5 mM MgCI2 (QIAGEN), 0.8 mM dNTP, 0.5 pmolIpL each of forward and
reverse primers, 2.5 U of HotStar Taq DNA polymerase (QIAGEN), and 0.5 pg of
A.tumefaciens genomic DNA. The reactions were incubated in a MasterGradient
thermocycler (Eppendorf) at 95•‹C for 15 min, then at 94•‹C for 1 min, 62•‹C for 30
sec, and 72•‹C for 1 min. The last 3 incubations were repeated for 50 cycles. The
final extension step was carried out at 72•‹C for 10 min. The amplified fragments
were cloned into the pCR2.1-TOP0 vector using the TOP0 TA Cloning Kit
(Invitrogen). The recombinant plasmids were transformed into TOP1 OF'
chemically competent E.coli cells. Ligations and transformations were performed
using materials and instructions provided by Invitrogen. The transformed cells
were grown in Luria-Bertani (LB) media supplemented with 100 pg1mL ampicillin
and plasmids were purified using QIAGEN Plasmid Miniprep kit.
The inserts containing A.fumefaciens CsaA gene were excised from the
TOP0 vector using restriction enzymes Ndel and Hindlll. The inserts were
subcloned into the PET-28a(+) overexpression vector (Novagen) designed to
express the protein with an N-terminal hexahistidine tag, and transformed into
E.coli BL21 (DE3) cells. The cloning procedure involved the same materials and
procedures as described in section 2.2.1, except that the restriction enzymes
Ndel and Hindlll were used to digest the vector. The resulting AtCsaNpET28a
construct sequenced at the UBC NAPS Sequencing facility using the universal
T7 promoter primer. The sequencing results were identical to the annotated entry
for A.fumefaciens CsaA.
The construct X15-AtCsaA was designed to express a 15-residue long
phage display derived peptide (VPGQKQHYVQPTAAN) at the N-terminus of
AtCsaA protein. For the X I 5-AtCsaA construct, primers XIS-sense: 5' gc ggc agc
CAT ATG gtt cct naq caa aas can cat tat qtt can ccn acs qca nct aat agc ggc gaa
att tc and T7-terminator 5' tat gct agt tat tgc tca g were used. Primer X15-sense
has Ndel restriction site at its 5' end (in capital letters), followed by the DNA
sequence encoding the phage display derived peptide (underlined), followed by
an overlapping region for annealing to the csaA gene. PCR was performed using
the AtCsaNpET28a construct as template. The new XIS-AtCsaA PCR product
was cloned into the vector pET28a(+) as described above, to create the plasmid
X I 5.1-CsaA-pET28.
3.2.2. Overexpression and Purification of AtCsaA and X I 5-AtCsaA
To check for AtCsaA overexpression, several BL2 1 (DE3) transformants
were grown in LB media supplemented with 50 pglmL kanamycin at 37•‹C for 4
hours, and then induced with 0.5 mM IPTG at 37•‹C for 2 hours. The pelleted
cells were lysed with addition of the lysis buffer (50 mM Tris-HCI pH8.0, 100 mM
NaCI, 40 pg/mL lysozyme, 1.8UIpL DNase) on ice for 30 min, and analyzed on
15% SDS-PAGE. All polyacrylamide gels used in SDS-PAGE were prepared
according to the recipe in Sambrook et all 1989. The protein expression was
optimized for length of induction and IPTG concentration.
For large scale protein overexpression, the same protocol as described in
section 2.2.2 was used, with the following modifications. 20 mL overnight culture
per 1 L of media were used to seed the cultures used for protein overexpression.
Cells were grown until the OD600 reading reached 0.5. The culture was induced
for protein overexpression with 0.5 mM IPTG at 37•‹C for 2 hours. The cell pellets
from each l L of culture were resuspended in 40 mL of 50 mM Tris pH 8.0, 0.3 M
NaCI. The resuspended cells were lysed in the French Pressure cell at 1000 psi
by passing through the cell 6 times. Prior to applying to Ni-NTA column, the
clarified cell supernatant was supplemented with 10 mM imidazole.
The cleavage of the His tag off AtCsaA by thrombin protease was
optimized by incubating AtCsaA with 5 and 1 units of thrombin per mg of protein
at 4•‹C and taking the aliquots at several different time points. After thrombin
cleavage, AtCsaA protein was applied to a Sephacryl S-100 HiPrep 26/60
column on an AKTA Prime system (Pharmacia Biotech), and run at 1 mL/min
93
with a buffer containing 20 mM Tris-HCI pH 8.0, 0.1 NaCI. Fractions containing
pure AtCsaA, as analyzed by SDS-PAGE, were concentrated for crystallization
using an Amicon ultra-centrifuge filter (Millipore). The protein concentration was
determined by the bicinchoninic acid (BCA) protein assay (Pierce).
For the Xl5-AtCsaA construct, a similar protocol was utilized to
overexpress, purify, and cleave the His tag off the protein, except that cells were
lysed in Avestin Emuliflex-3C cell homogenizer.
3.2.3. Crystallization and Data Collection of AtCsaA and X I 5-AtCsaA
In order to obtain crystals of AtCsaA, initial crystallization trials were
carried out using sparse matrix crystal screens (Hampton research). Initial
crystals were obtained from an aliquot of His-tagged AtCsaA by hanging drop
vapour diffusion method; the drops included 0.5 pL of 20 mg/mL AtCsaA protein
and 0.5 pL of reservoir solution and were hanging over 1 mL of reservoir solution.
Grid screens with varying pH, precipitant, and additive conditions were employed
to refine the initial crystallization conditions. To improve the quality of the
crystals, the hexahistidine tag was cleaved off the proteins with thrombin. For
subsequent experiments, sitting drop vapour diffusion at room temperature was
used to crystallize AtCsaA, and the drops consisted of 1 pl protein (13 mglml)
and 1 pl reservoir solution.
The Hampton research crystal screens were also used to obtain crystals
of X15-AtCsaA. To improve crystallization, His tag was cleaved off XIS-AtCsaA
with thrombin protease. Crystals of X15-AtCsaA were produced by the sitting
drop vapour diffusion technique at 19•‹C. Drops consisted of 1 pl protein (9
mglml) and 1 pl reservoir solution.
Prior to data collection, AtCsaA crystals were transferred into a
cryoprotectant solution that contained the mother liquor in which 15% of water
was replaced with ethylene glycol. For the crystals of X15-AtCaA, mother liquor
was used as a cryoprotectant. The diffraction data were collected at the Simon
Fraser University Macromolecular X-ray Diffraction Data Collection Facility using
a RAXlS IV++ image plate detector mounted on a 007 Rigaku X-ray generator
with VariMax CuHF optics. Data for AtCsaA and X15-AtCsaA crystals were
collected with a crystal-to-detector distance of 120 mm and 150 mm,
respectively. Data were collected at 100K using an X-stream 2000 cryo-system.
A total of 192 and 186 frames were collected for AtCsaA and X15-AtCsaA,
respectively, using 0.5O oscillations. Each image was exposed for two minutes.
Data were indexed, integrated and scaled with the program Crystal Clear
(Pflugrath, 1999).
3.2.4. Structure Determination and Refinement of AtCsaA and X I 5-AtCsaA
The structures of AtCsaA and X15-AtCsaA were solved by molecular
replacement with the program Phaserl.2 (McCoy et all 2005). The search model
used was a homology model of AtCsaA constructed with CPH (Lund et al, 2002)
based on coordinates from TtCsaA (PDB 1DG7, chain A) (Kawaguchi et all
2001). The structures were refined using restrained refinement in REFMAC5
(Murshudov et al, 1997) and simulated annealing, energy minimization and B-
factor refinement in CNS (Brunger et al, 1998). Manual adjustments to the atomic
coordinates were performed with the program Coot (Emsley & Cowtan, 2004).
The final models were obtained by running restrained refinement in REFMAC5
with TLS restraints obtained from the TLS motion determination server (Painter &
Merritt, 2006). Refinement statistics are shown in Table 3.2. The final refined
structures of AtCsaA and X15-AtCsaA atomic coordinates were deposited at the
Protein Data Bank, with accession numbers 2Q21 and 2Q2H, respectively.
3.2.5. Structural Analysis
The program PROCHECK (Morris et al, 1992) was used to analyze the
quality of the final refined model. The program Superpose (Maiti et al, 2004) was
used for superimposition of CsaA structures. The program CASTp (Binkowski et
al, 2003) was used to analyze the surface of CsaA. The hydrogen bonds were
determined with WhatlF server's optimal hydrogen bonding network (Hooft et all
1996). Intermolecular interactions were measured using the protein-protein
interaction server (Jones & Thornton, 1995). Figures were prepared with PyMol
(DeLano, 2002).
3.3. Results and Discussion
3.3.1. PCR and Cloning of AtCsaA
Figure 3.1 PCR amplification of A.tumefaciens CsaA gene.
PCR products were separated on 1% agarose gel, stained with ethidium bromide and visualized under UV light at 320nm.
PCR successfully amplified a region of genomic A.tumefaciens DNA
corresponding to the size of the annotated csaA gene. PCR worked very well, as
no non-specific products could by detected. (Figure 3.1).
CsaA - #1 #2 2
Figure 3.2 Cloning of A.tumefaciens CsaA
A) AtCsaAITopo construct purified from 2 TOPIOF' transformants and digested with restriction enzymes Ndel and Hindlll to liberate the insert. B) AtCsaAIpET28a constructs purified from 6 Novablue transformants and digested with Ndel and Hindlll to liberate the insert. Reactions were separated on a 1% agarose gel. The location of the bands corresponding to liberated A.tumefaciens csaA insert is indicated with arrows.
Cloning of PCR-amplified A.tumefaciens csaA gene into the pCR2.1-
TOP0 vector and subloning into the pET28a(+) vector was also successful. The
restriction enzyme digest of the constructs liberated bands similar in size to the
csaA gene (342 b.p.) (Figure 3.2).
3.3.2. Overexpression and Purification of AtCsaA
Figure 3.3 Purification of AtCsaA by nickel affinity chromatography.
The fractions obtained by nickel affinity chromatography were analyzed for protein content on 15% SDS-PAGE stained with Coomassie Blue. Std: broad range standard; cell supt: supernatant obtained after centrifugation of lysed cells. The arrow indicates the bands corresponding to AtCsaA protein (14.4 kDa).
The purification of His-tagged AtCsaA protein by nickel affinity
chromatography was successful because the protein bound to the Ni-NTA beads
in large quantities, whereas most of the contaminating proteins were removed in
,the flowthrough from the column (Figure 3.3). Most of AtCsaA protein eluted in
the fractions containing 200-400 mM imidazole and was sufficiently pure to carry
out initial crystallization experiments.
5U thrombin I mg AtCsaA 1 U thrombin / mg AtCsaA
1 2 4 1 6 1 1 2 4 1 6 ) . hours mubation of
Figure 3.4 Optimization of the thrombin digest reaction of A.tumefaciens CsaA.
AtCsaA protein was incubated at 4•‹C with 5 and 1 units of thrombin per mg of AtCsaA. Aliquots were taken at 1, 2, 4, and 16 hours, and reaction was quenched with addition of SDS-PAGE loading buffer. Proteins were separated on 20% SDS- PAGE and stained with Coomassie Blue.
The thrombin cleavage reaction to remove the His tag off the AtCsaA
protein was optimized using different thrombin concentration and time of
reaction. Complete cleavage was achieved when 5 units of thrombin per mg of
AtCsaA protein were added and the reaction was incubated at 4•‹C for 16 hours
(Figure 3.4).
Crystallization of AtCsaA and XIS-AtCsaA
0.1 M HEPES pH 7.5, 2% V/V PEG 400, 1.8M ammonium sulfate
0.2 M MgOAc, 0.1 M Sodium Cacodylate pH 6.5, 22% PEG 6000.
Figure 3.5 Initial crystals of AtCsaA.
A and 6) lnitial crystals produced from AtCsaA with intact His tag. C) A diffraction pattern produced by the crystal depicted in pane B.
Initial crystals of AtCsaA were obtained from Crystal Screen 1 (Hampton
Research). Methods such as varying the pH, concentration of the precipitant, and
using different additives were employed to optimize the crystallization conditions.
"Needle" crystals of AtCsaA formed in 0.1 M HEPES pH 7.3-7.7, 2% vlv
PEG 400, 1.6-2.OM ammonium sulfate (Figure 3.5A). Protein concentration was
20 mglml, in 50 mM Tris-HCI pH 8.0. First crystals appeared after 2 days of
incubation at room temperature. Removing the additive or changing it to ethanol,
glycerol, or ethylene glycol resulted in formation of bigger but more irregular
crystals.
Large crystals of a distinctive form with pointy tip, sharp facets, and flared
tails formed in 0.2 M MgOAc, 0.1 M Sodium Cacodylate pH 6.3-6.5, 20-21% wlv
PEG 8000 or 22-24% wlv PEG 6000 (Figure 3.58). Protein concentration was 20
mglml in 50 mM Tris-HCI pH 8.0, 0.1 M NaCI. First crystals appeared after 5
days of incubation at room temperature. These crystals diffracted to 2.1A at UBC
X-ray diffraction facility and to 1.5 A at the Advanced Light Source, Lawrence
Berkeley National Laboratory, University of California at Berkeley.
In order to improve AtCsaA and X15-AtCsaA crystals, thrombin protease
was used to cleave the His tag off these proteins. Crystals of AtCsaA with the His
tag cleaved off were obtained at room temperature in 1.8M ammonium sulfate,
0.1M HEPES pH 7.5, 2% PEG400, and 5% ethylene glycol. Crystals of X15-
AtCsaA with the His tag cleaved off were obtained in 30% PEG4000, 0.4M
NH~OAC, and 0.1M Na-citrate pH 5.5. These crystals were used for data
collection and structure determination. AtCsaA crystals diffracted to beyond 1.55
A resolution, whereas X15-AtCsaA crystals diffracted to beyond 1.65 A
resolution. Data collection statistics can be found in Table 3.1
Table 3.1 The data collection statistics for the structures of AtCsaA and XIS-AtCsaA.
Data Collection AtCsaA X I 5-AtCsaA
Crystallization conditions 1.8M ammonium sulfate, 30% Peg4000, 0.4M 0.1M HEPES pH 7.5, 2% NH40Ac, 0.1M Na-citrate Peg400, and 5% ethylene pH 5.5. glycol
Molecular weight (of dimer) 24,893 Da 28,246 Da Space group p61 p61 a, b, c, (A) 60.6 x 60.6 x 1 13.4 60.5 x 60.5 x 115.3 Molecules in ASU 2 2 Resolution (A) 52.53-1.55 (1.61- 1.55) 23.86 - 1.65 (1.71-1.65) Total observed reflections 3691 54 146729 Unique reflections 341 23 25681 % completeness 99.8 (99.6) 89.4 (49.7) I 1 o(l) 23.7 (7.2) 30.0 (8.6) Rrnerge (%I# 4.0 (34.1) 3.9 (16.2) Redundancy 10.8 (10.1) 5.7 (4.5)
Values in parentheses are for the highest resolution shell.
i R ~ r g e = ~ I 1 - ( l ) l I ~ ( z ) , where I is the observed intensity obtained from multiple observations of symmetry-related reflections after rejections.
3.3.4. Structure Determination and Refinement of AtCsaA and X I 5-AtCsaA
Structures of AtCsaA and X15-AtCsaA were solved by molecular
replacement, using T.thermophi1u.s CsaA structure (PDB ID lgd7) as a model.
Despite the different crystallization conditions, both AtCsaA and X15-AtCsaA
proteins produced crystals in the space group P6,, with 1 CsaA dimer in the
asymmetric unit. Interestingly, the unit cell dimensions were somewhat different,
with X15-AtCsaA unit cell being about 2 A larger at the c edge.
In order to improve the agreement between the models and the
experimental data, the datasets for AtCsaA and X15-AtCsaA were re-scaled and
averaged to resolution cut-offs of 1.55 A and 1.65 A, respectively. The models
were further improved by manual adjustment of the positions of amino acid side
chains and modeling in the solvent molecules, such as water, ethylene glycol,
citrate and sulfate ions. The refinement progress is shown in Table 3.2. The
sulfate ion was particularly important as it was found residing in the substrate
binding site in the AtCsaA structure without a peptide. (discussed in section
3.3.7). The final refinement statistics can be found in Table 3.3.
The final refined structure of AtCsaA includes all residues, except for
residues 29-31 in chain B and the N-terminal methionine in each chain, which
were not modeled due to lack of density. The residue Glu28 in chain B was
modeled as alanine due to lack of side chain density. In the structure of X15-
AtCsaA, clear electron density was obtained for the last 5 residues of the peptide
tethered at the N-terminus of molecule A (Q-6P-5T-4A3A2N-1). NO difference
density was observed for the peptide at the N-terminus of molecule B, where the
first residue seen is Gly3. Electron density was also missing for a loop region in
molecule B (residues 27-31) and the side chain of Glu43 in molecule A.
Table 3.2 The progress of refinement of AtCsaA and X15-AtCsaA structures
I 1 AtCsaA I Xl5-CsaA I
initial
final 1 0.178 1 0.208 / 0.161
Rwork
0.220
0.173
Rfree
0.245
Rwork
0.183
R h e e
0.213
Table 3.3 The refinement statistics for the structures of AtCsaA and XIS-AtCsaA.
Refinement AtCsaA X1 5-AtCsaA
Protein residues Waters Other solvent molecules
Rwork (%) Rfree (%) r.m.s. deviations Bonds (a) Angles (")
Overall B (a2) (all atoms) (protein) +I++
(peptide) (solvent)
Ramachandran "' (%)
Rwfi = ZIIFOI-IF~~~/ZIF~I SRfree is calculated the same way as R factor for data omitted from refinement (5% of reflections for all data sets). +Protein B-factor for chain A. ++ Protein B-factor for chain B. *Residues in the most favorable region **Residues in additionally allowed region
3.3.5. An Overview of the AtCsaA structure and comparison to BsCsaA
The structure of CsaA from A.tumefaciens is very similar to the structures
of CsaA from B.subtilis and T.thermophillus. It is a homodimeric molecule, in
which each monomer is 11 3 amino acids long and consists of 2 a-helices and 10
P-strands (Figure 3.6A). Strands PI, P2, P3, P4, P7, and helix a2 form the core of
the monomer, an oligonucleotide 1 oligosaccharide binding (08) fold. The N-
terminal helix a1 and the C-terminal strands P8 and P9 contain the majority of
residues that participate in formation of interchain hydrogen bonds important for
dimerization. The r.m.s.d. of superposition of dimeric AtCsaA structure and CsaA
from B.subtilis (2NZ0, chains AB) is 1.68 A over 218 a-carbons, and that of
AtCsaA and CsaA from T.thermophilus (1GD7, chains AB) is of 1.77 A over 216
a-carbons. The three structures superimpose very well except for several flexible
loop regions that deviate in position (indicated in Figure 3.6B). Like BsCsaA and
TtCsaA, AtCsaA contains two large cavities (one in each monomer) that are
separated by a rotation of approximately 90 degrees about the axis of the dimer
(Figure 3.6C). These cavities are putative substrate binding sites and are
composed of residues 26, 28-31, 33, 49-53, 73-76, 78, 83-90, 97, and residues 4'
and 11 3' from adjacent monomer. The majority of these residues are located on
the flexible loops described above, indicating that the binding site may be
dynamic. Most of the residues that make up the putative binding sites are
conserved between AtCsaA and BsCsaA. It is interesting to note that the pattern
of strong positive and negative electrostatic potential in the vicinity of the binding
site, as described for the structures of BsCsaA and TtCsaA in section 2.3.9, also
occurs in AtCsaA (Figure 3.6C). Due to high sequence variability in that region,
the position of the negatively charged area is somewhat different in all three
proteins, however, the overall tendency of having both negatively and positively
charged regions in the vicinity of the binding cavity is preserved.
res. 9
Figure 3.6 The structure of AtCsaA
A) A cartoon diagram of dimeric AtCsaA. Molecule A is in green and Molecule B is in pink. B) A ribbon diagram of the superimposed structures of AtCsaA (green), B.subtilis CsaA (2NZH, red), and T.thermophilus CsaA (1GD7, blue). C) A surface representation of the AtCsaA structure coloured according to the negative (red), positive (blue), or neutral (white) electrostatic potential. Location of the putative binding sites is indicated with arrows.
Table 3.4 The interchain hydrogen bonds between the two monomers of AtCsaA.
Donors Acceptors
Residue Atom Residue Atom
'ser 2A OG Glu 118 0
'ser 28 N Asp 14A OD2
Ile 5 N Asn 72' 0
' ~ y s 12B NZ Asp 9A OD2
Tyr 57 OH Glu 103' OEl
Asn 72 ND2 Gly 3' 0
Gly 89 N Cys 1 13' 0
Ala 101 N Ala 101' 0
Glu 103 N Leu 99' 0
' ~ r g 1048 NH1 His 56A ND1
Cys 113 N Gly 89' 0
Due to a local two-fold axis of symmetry in the dimer, the same residues form hydrogen bonds in both chains of the dimer (except as noted). Bold font indicates bonds that are conserved between AtCsaA and BsCsaA. # occurs only once per dimer
The AtCsaA dimer is held together by 18 hydrogen bonds, 14 of which are
formed by the same residues in each monomer due to the two-fold symmetry of
the dimer. The remaining 4 bonds occur only once in each dimer. The pattern of
interchain hydrogen bonding is well conserved between the structures of AtCsaA
and BsCsaA. The majority of main-chain hydrogen bonds occurs between
residues that occupy equivalent positions in sequences of AtCsaA and BsCsaA.
Two side-chain mediated hydrogen bonds are also conserved in the structures of
AtCsaA and BsCsaA and occur between the donor-acceptor pairs Tyr 57-Glu 103'
and Lysl2-Asp9'. These residues may therefore be important for dimerization of
the CsaA protein.
3.3.6. Interaction of XIS-AtCsaA with the co-crystallized peptide
The crystal structure of X15-AtCsaA reveals that the N-terminally fused
peptide from chain A of the dimer binds into a large pocket of chain A in a
symmetry related dimer. Six of the fifteen peptide residues (Q-6P-5T-A3A2N-1) in
chain A are represented by a well-defined electron density and interact with the
residues of the pocket in a symmetry related dimer. The electron density is
missing for the remainder of the peptide in chain A and for the entire peptide in
chain B. This is most likely due to a great degree of thermal motion in these
residues as they are not stabilized by interactions with other protein residues.
The peptide binds into the AtCsaA pocket in an extended conformation
and buries 446 A2 of the AtCsaA surface in the peptide-protein interface. This
surface is 58.2% non-polar in nature and consists of residues 26, 28-31, 33, 50-
52, 76, 78-80, 86, 87 from chain A and residue 113 from chain B, which contact
the peptide through both polar and non-polar interactions. There are 7 hydrogen
bonds between the atoms of the peptide and the pocket (Table 3.5). Two of
these hydrogen bonds are strictly main chain interactions and the other five are
side chain mediated. In addition to the bonds listed in Table 3.5, there is an
indirect hydrogen bond between the peptide and the pocket, which occurs via a
water molecule. The water W24 is coordinated in its position via hydrogen bonds
to the peptide atom Glen (-6) OEl and the pocket atoms Thr87A N, Ser 50A 0,
and Ser49A OG. All pocket residues that participate in hydrogen bonding
interactions do so through main chain atoms, with the exception of Arg76A. The
guanidinium group of this residue forms a bifurcated hydrogen bond to the main
Figure 3.7 The structure of AtCsaA in complex with a phage-display derived peptide (XI 5-AtCsaA).
A) A cartoon representation of the X15-AtCsaA structure. The phage-displayed peptide at the N-terminus of chain A binds into the pocket of chain A of a symmetry related dimer The two dimers are coloured green and yellow. B) A surface representation of the view in (A). Nitrogens are in blue, oxygens in red and carbons in green or yellow. C) A close-up view of the region enclosed in a box in pane (B), showing the interactions between the pocket and the peptide. Peptide residues are shown as yellow sticks, and protein residues that make contact with the peptide as green sticks. Hydrogen bonds are shown as red dashes. Peptide and protein residues are labelled in red and black, respectively.
Table 3.5 Direct hydrogen bonds between the peptide and XIS-AtCsaA.
Peptide XI 5-AtCsaA Bond
Residue Atom Residue Atom Length (A) Gln (-6) N E2 Thr 87A 0 3.1
Gln (-6) NE2 Cys 113B OXT 2.7
Gln (-6) NE2 Cys113B 0 3.4
Pro (-5) 0 Arg 76A NH1 3.0
Pro (-5) 0 Arg 76A NH2 3.2
Th r (-4) 0 Arg 30A N 2.8
Ala (-2) N Glu 28A 0 2.5
chain oxygen on the peptide residue Pro(-5).
The pocket can fit 4 peptide residues, since Asn (-1) and Ala (-2) do not
enter the pocket. Three of these residues (Gln (-6), Pro (-5), and Ala (-3)) point
down, towards the pocket and possibly act as specificity determinants for the
peptide. Gln (-6) is well coordinated in its position via three direct and one
indirect hydrogen bonds from its side chain atoms NEI and OEl to the pocket
atoms. Pro (-5) and Ala (-3) side chains interact with the pocket atoms through
non-polar contacts and are constricted in their positions by main chain hydrogen
bonds to AtCsaA. Having large residues in place of Pro(-5) and Ala (-3) in the
peptide would be disfavoured due to steric clash with the pocket atoms. Charged
residues would also likely be disfavoured due to the hydrophobic character of the
pocket at the sites of Pro(-5) and Ala (-6) binding.
Other chaperones that recognize and bind short motifs of sequence with
few specificity determinants have been characterized. For example, E.coli DnaK
chaperone was co-crystallized with a 7-residue phage display-derived peptide
which binds into a substrate binding groove in DnaK in an extended conformation
((Zhu et al, 1996), discussed in section 1.4). A leucine residue from the peptide is
buried in a deep hydrophobic pocket in DnaK and acts as a key specificity
determinant for the chaperone-peptide interaction The peptide interacts with
DnaK through side-chain mediated non-polar contacts and main-chain hydrogen
bonds. Similar chaperone-peptide interactions were described for Ydjl, an hsp40
homologue from yeast ((Li et all 2003), discussed in section 1.4). SurA, a
periplasmic chaperone, was co-crystallized with a symmetry related peptide in
the substrate binding site ((Bitto & McKay, 2002), discussed in section 1.3). In
this case, the peptide adopts an a-helical conformation and contains a leucine
and a valine residues that act as specificity determinants by interacting with the
hydrophobic pockets in the chaperone. The interactions of AtCsaA with the
peptide are similar to those described above in that AtCsaA binds to a short, 4-
residue motif in the peptide mostly through main-chain hydrogen bonding and
van der Waals interactions. Specificity likely arises from having a glutamine
residue at position (-6) in the peptide, and small uncharged residues at positions
(-5) and (-3). It is therefore possible that CsaA can bind a wide range of
substrates that display these characteristics.
3.3.7. A comparison of the substrate binding pockets in the structures of CsaA from AAumefaciens, B.subtilis, and T. thermophilus.
The position of the pocket residue Arg76 appears to be the key
determinant of the pocket's ability to bind the peptide. In molecule A, which has
the peptide bound, the guanidinium group of the Arg76 side chain points towards
the pocket, and makes a bifurcated hydrogen bond with the carbonyl oxygen of
peptide residue Pro(-5). In molecule B, which lacks the bound peptide, Arg76
points away from the pocket and towards the solvent. Interestingly, in the
structure of AtCsaA, which was crystallized without a peptide, Arg76 also points
towards the pocket in molecule A and towards the solvent in molecule B (Figure
3.8). A close examination of the pocket in molecule A in the AtCsaA structure
without a peptide revealed the presence of a sulfate ion occupying the same
position in the pocket as the peptide residue Pro(-5). Arg76 makes hydrogen
bonds to the oxygen atoms on this sulfate, similar to the interactions of Arg76
and the peptide Pro (-5) 0 in the structure with the peptide. In addition, the
sulfate ion is highly coordinated in its position via direct and indirect hydrogen
bonds to other residues of the pocket. Both the structures with and without the
peptide were crystallized in the space group P61, and with similar unit cell
dimensions. In this particular space group, steric hindrance from the symmetry
related atoms restricts the thermal motion of Arg76 from molecule A and
stabilizes it in the "down" position, which leads to narrowing of the binding pocket
and formation of stable hydrogen bonding interactions with the peptide in the
crystal. Since Arg76 sits on a solvent exposed loop, it is likely that in vitro and in
vivo, Arg76 has a much greater degree of freedom of movement, allowing it to
Pocket A
AtCsaA Pocket A
BsCsaA --
Pocket B
Pocket B
TtCsaA
Figure 3.8 The substrate binding pockets from the structures of AtCsaA, XIS-AtCsaA, BsCsaA, and TtCsaA.
Surface representations of the substrate binding pockets from the structures of X15- AtCsaA (PDB ID 2Q2H), AtCsaA (2Q21), BsCsaA (2NZO_AB), TtCsaA (1 GD7-AB). Residues important for protein-peptide interactions are shown as sticks.
move "up" and "down" and to transiently interact with and stabilize the peptide
bound into the pocket of CsaA. In the previously published structures of CsaA
from B.subtilis and T.thermophilus, an arginine and a lysine residues,
respectively, occupy positions equivalent to Arg76 in the structure of
A.tumefaciens CsaA. These residues lack a complete side chain density and are
likely flexible due to a greater degree of thermal motion. Moreover, Arg76 is
highly conserved and all CsaA proteins contain either a lysine or an arginine at
that position (Figure 2.10, alignment). This highlights the role for Arg76 as a
clamp which moves down to transiently lock and stabilize the peptide when it is
bound into the substrate binding pocket of CsaA.
The peptide atom Gln (-6) NEI makes hydrogen bonds to three main
chain atoms in the pocket (Thr87A 0, Cysl l3B OXT, and Cysl l3B 0 ) that
together make up a patch of negative charge in the floor of the peptide binding
cavity. Comparison of the substrate binding sites of CsaA structures from
A.tumefaciens, B.subtilis, and T.thermophilus reveals that the position of this
patch of negative charge is conserved among all three structures and is formed
by main chain atoms from residues that occupy equivalent positions in the
sequence (Figure 3.8). This conserved patch of negative charge in the pocket
may act as another determinant of the chaperone-peptide interaction by
providing a possibility of hydrogen bonds between the pocket and a proton donor
atom in the peptide.
3.4. Conclusion
The structure of AtCsaA in complex with a phage display-derived peptide
provides an insight into the mode of the CsaA binding to its substrates. CsaA
binds four peptide residues into an open pocket on its surface through hydrogen
bonds and van der Waals contacts. Three residues of the bound peptide might
act as specificity determinants for the chaperone-peptide interactions. Gln (-6)
forms side-chain mediated hydrogen bonds to three main chain oxygen atoms in
the pocket. These main chain oxygen atoms form a patch of negative surface
potential that is well conserved in the structures of CsaA from A.tumefaciens,
B.subtilis, and T.thermophilus, thus providing a possibility of hydrogen bonds
between the pocket and a proton donor atom in the peptide. In addition, small
uncharged residues are required at positions (-5) and (-3) of the peptide to avoid
steric clashes with the pocket atoms. The well-conserved pocket residue Arg76
acts as an important selectivity determinant for the chaperone. When the peptide
is bound, the side chain of Arg76 moves down to transiently interact with and
stabilize the peptide by forming hydrogen bonds from its guanidinium group to a
main chain oxygen on the peptide. Since AtCsaA-peptide interactions have
limited specificity, CsaA is likely to bind a wide variety of substrates thus acting
as a general chaperone.
Biochemical studies would be required in order to confirm the information
derived from the structural data on CsaA. For example, isothermal titration
calorimetry or surface plasmon resonance spectroscopy could be employed to
examine the kinetics of the phage display derived peptide binding to CsaA. In
addition, biological substrates for CsaA need to be identified in order to elucidate
its role in the Sec-dependent protein secretion.
APPENDIX A. CLONING, PURIFICATION, AND CRYSTALLIZATION OF A. TUMEFACIENS SECB
A.1. Introduction
The homotetrameric protein SecB functions as a targeting factor and a
chaperone in the Sec-dependent translocation system (discussed in section
1.12). SecB acts post-translationally, binding to the core regions of the newly
synthesized proteins and targeting them to the SecA subunit of the Sec
translocase for insertion or translocation across the membrane (Driessen et al,
2001). At the same time, SecB protects these proteins from misfolding and
aggregation. Although SecB and CsaA share no sequence or structural similarity,
they were proposed to fulfill the same function in the Sec-dependent protein
translocation and even to have an overlapping substrate specificity (Linde et all
2003). In addition, CsaA was proposed to substitute for SecB in species such as
Bacillus subtilis, which lack SecB. We chose to carry out crystallographic studies
of SecB and CsaA from A.tumefaciens simultaneously to compare the structural
features of each protein that might allow to determine the individual roles of SecB
and CsaA in Sec-dependent translocation.
A.2. Materials and Methods
A.2.1. PCR and Cloning
A sequence corresponding to secB gene was amplified by PCR from
A.tumefaciens genomic DNA. The primers for PCR reaction were designed
based on a Swiss-Prot annotated sequence with accession number Q8UJC2.
PCR was carried out with the forward primer 5' CAT ATG acc gct gaa aat ggc
gca cag ggc gca, incorporating Ndel restriction site (in capital letters), and the
reverse primer 5' AAG CTT tta gtt cgg gac agc ctg aac ctg ggc ctt, incorporating
Hindlll restriction site. PCR reaction conditions were identical to those described
for csaA from A.tumefaciens in section 2.2.1, except PCR was carried out for 43
cycles. The amplified fragments were cloned into the pCR2.1-TOP0 vector using
the TOPO TA Cloning Kit (Invitrogen). The recombinant plasmids were
transformed into TOPIOF' chemically competent E.coli cells. Ligations and
transformations were performed using materials and instructions provided by
Invitrogen. The transformed cells were grown in Luria-Bertani (LB) media
supplemented with 100 pglmL ampicillin and plasmids were purified using
QIAGEN Plasmid Miniprep kit.
The inserts containing A.tumefaciens secB gene were excised from the
TOPO vector using restriction enzymes Ndel and Hindlll, subcloned into the
PET-28a(+) overexpression vector (Novagen), and transformed into E.coli
BL21(DE3) cells using the same materials and procedures as described in
section 3.2.1. The construct AtSecBIpET28a was sequenced at the UBC NAPS
Sequencing facility using the universal T7 promoter primer. The sequencing
results were identical to the annotated entry for A.tumefaciens SecB (AtSecB).
A.2.2. Protein Overexpression and Purification
The protein expression was optimized for length of induction and IPTG
concentration using the same procedure described for AtCsaA. The conditions of
119
protein overexpression and purification by ~ i * ' affinity chromatography were
identical to those described for AtCsaA in section 3.2.2. In order to improve
crystallization conditions for AtSecB, thrombin protease was utilized to cut the
hexahistidine tag off the protein. The protein was further purified by size
exclusion chromatography using HiPrep 16/60 Sephacryl S-100 HR size
exclusion column (Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCI
pH 8.0, 100 mM NaCI. Fractions containing AtSecB were pooled together and
concentrated to 10 mglmL using an Amicon ultra-centrifugal filter (Millipore).
A.2.3. Crystallization and Data Collection
The initial crystallization conditions were obtained with sparse matrix
crystal screens (Hampton Research) using hanging drop vapor diffusion method.
Drops included 0.5 pL of the reservoir solution and 0.5 pL of 10 mglml SecB and
were hanging over 1 mL reservoir solution. Initial crystals were obtained with
solution #41 from Crystal Screen I (Hampton Research), which contained 0.1M
HEPES-Na pH 7.5, 10% isopropanol, 18-20% polyethylene glycol (PEG) 8000.
Crystals were obtained after 7 days of incubation at room temperature. Grid
screens with varying pH and precipitant concentrations were employed to refine
the initial crystallization conditions. The crystallization conditions were further
optimized by varying the protein concentration, concentration and nature of the
additives, and incubation temperature. Larger crystals suitable for data collection
were obtained from an aliquot of SecB with His tag cleaved off. The reservoir
solution contained 0.2 M Mg2S04, 0.1 M Na Cacodylate pH 6.5, and 16-22%
PEG 8000. The drop contained 0.5 pL reservoir solution, 0.5 pL of 15 mglml
AtSecB protein, and 0.5 pL of 0.1% ~ l u g e n t ~ ~ detergent (Calbiochem). Crystals
appeared after two days of incubation at room temperature. An attempt to collect
data from these crystals was made at SFU Macromolecular X-ray Diffraction
Data Collection Facility using equipment and methods described in section 2.2.3.
A.3. Results and Discussion
A.3.l. PCR and Cloning
1 kb ladder SecB
ftl #3 - SecB inserts (483 b.p.)
.*
mass ruler - - 1 kbllOO ng . . , 700 bpl70 ng
500 b.pJ50 n-g
Figure A1 PCR and cloning of A.tumefaciens secB gene
A) PCR amplification of A.tumefaciens secB gene. B) AtSecBPTopo construct purified from 3 TOPIOF' transformants and digested with restriction enzymes Ndel and Hindlll to liberate the insert. C) AtSecBIpET28a constructs purified from 5 Novablue transformants and digested with Ndel and Hindlll to liberate the insert. The reaction products were separated on 1 % agarose gel.
PCR successfully amplified a region of genomic DNA from A.tumefaciens
corresponding to the annotated secB gene (Figure A1 A).The cloning of PCR-
amplified Ahmefaciens secB gene into the pCR2.1-TOP0 vector and subloning
into the pET28a(+) vector was successful as the restriction enzyme digest of the
constructs liberated bands similar in size to the secB gene (483 b.p.) (Figure A1
AB).
A.3.2. Overexpression and Purification of AtSecB protein
- - -
SecB tetramer (70 kDa)
SecB (higher MW aggregate)
Figure A2 Overexpression and purification of A.tumefaciens SecB.
A) Small scale overexpression of AtSecB. Cells were grown to an O.D. of -0.5 and induced with 0.5 mM IPTG at 37•‹C for the specified length of time. Cell lysates were separated on 15% SDS-PAGE. B) 15% SDS-PAGE of the SecB fractions obtained during purification by ~ i ' ' affinity chromatography. All gels used in SDS-PAGE were stained with Coomassie Blue. Bands corresponding to SecB are identified by arrows. C) Cleavage of the His tag off SecB by thrombin. Reactions were incubated at 4•‹C for the specified length of time and separated on 20% SDS-PAGE. D) The profile of SecB elution from the HiPrep 16160 Sephacryl S-100 HR size exclusion column (Amersharn Biosciences).
The small scale induction was successful, as the cells induced with IPTG
showed a very large band on the SDS-PAGE, which corresponds to -20 kDa,
similar to the size of a monomer of His-tagged AtSecB (Figure A2 A). The
optimal amount of AtSecB was produced after 2 hours of induction with 0.5 mM
IPTG at 37•‹C. Purification of His-tagged AtSecB by ~ i ~ ' affinity chromatography
was very efficient, as the eluted protein was very pure (Figure A2 B). Most of the
AtSecB eluted in fractions containing 200-400 mM imidazole. Thrombin cleavage
was used to remove the His tag from AtSecB. The reaction was optimized by
incubating AtSecB with 2 different concentrations of thrombin protease at 4•‹C for
1-16 hours (Figure A2 C). Cleavage was complete when 5 units of thrombin were
used per mg of AtSecB, and reaction was incubated at 4•‹C for 16 hours. In order
to separate the cleaved AtSecB from thrombin and other contaminants, an
aliquot of AtSecB was subjected to size exclusion chromatography. The size
exclusion profile showed two peaks, the larger of which corresponds to SecB
tetramer (Figure A2 D). The smaller peak corresponds to a higher molecular
weight aggregate of monomeric SecB, as confirmed by SDS-PAGE. Fractions
corresponding to tetrameric SecB were collected, concentrated, and used in
crystallization experiments.
A.3.3. Crystallization and Data Collection
A initial crystals B optimized crystals
AtSecB with His tag 0.1 M HEPES pH 7.5
10% isopropanol 18-20% PEG 8000
AtSecB without His tag 0.2 M Mg2S04
0.1 M Sodium cacodylate pH 6.5 16-22% PEG 8000
(+ 0.033% ElugentTM in the drop)
Figure A3 Crystals of A.tumefaciens SecB.
A and B) Initial and optimized crystals of AtSecB. Crystallization conditions are listed below each picture.
Initial crystals of AtSecB with His tag intact were obtained using Hampton
Research Crystal Screens. However, those crystals were too small to be used in
diffraction experiments. Optimizing crystallization conditions failed to produce
bigger crystals. In order to improve the size and quality of crystals, AtSecB with
His tag cleaved off was used in further crystallization experiments. After
extensive screening of crystallization conditions, crystals depicted in Figure A3
were obtained. These crystals looked sufficiently large and regular to be used in
diffraction experiments. However, these crystals produced no diffraction pattern.
This might be due to the fact that crystals adhered to the bottom of the sitting
drop pedestal on which they grew and were extremely difficult to remove due to
their fragility. Thus, crystals most likely were damaged during transfer to the cry0
solution. To overcome this problem, it might be necessary to use crystallization
setups other than sitting drop vapour diffusion to prevent AtSecB crystals
adhering to surfaces. Another possibility that might explain lack of diffraction from
AtSecB crystals is that the cry0 solution was not optimal and caused damage to
the crystals. If that is the case, then the cry0 solution can be optimized by
changing the nature and concentration of cryoprotectants.
APPENDIX B.
Figure 82 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P&21.
Figure B3 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P42,2.
131
Figure 64 Ramachandran plots of the crystallographic models of AtCsaA ligand-free (A) and with the symmetry related in the putative binding site (B) .
1 U9C
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earo
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hi
lus
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rmot
oga
mar
itim
a
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char
omyc
es
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ae
Xen
opus
lae
vis
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Hom
o sa
pien
s
Xen
opus
laev
is
Ho
mo
sap
iens
Bor
ek,
D.,
et a
l. S
truc
tura
l ana
lysi
s of
DJI
sup
erfa
mily
. T
o b
e p
ublis
hed.
Join
t C
ente
r fo
r S
truc
tura
l Gen
omic
s (J
CS
G)
Cry
stal
st
ruct
ure
of a
rche
ase,
pos
sibl
e ch
aper
one
(TM
108
3)
from
The
rmot
oga
mar
itim
a at
2.0
A r
esol
utio
n. T
o be
pu
blis
hed.
Dag
anzo
, S
.M.,
et a
l. S
truc
ture
and
Fun
ctio
n of
the
Con
serv
ed C
ore
of H
isto
ne D
epos
ition
Pro
tein
As
fl .
Cur
r.B
iol.
2003
. v1
3 p2
148
Eng
lish,
C.M
., et
al.
Str
uctu
ral b
asis
for
the
hist
one
chap
eron
e ac
tivity
of
as
fl .
Cel
l 200
6. v
127
p495
Eng
lish,
C.M
., et
al.
Str
uctu
ral b
asis
for
the
hist
one
chap
eron
e ac
tivity
of
asf
l . C
ell 2
006.
v12
7 p4
95
Ant
czak
, A
.J.,
et a
l. S
truc
ture
of t
he y
east
his
tone
H3
- A
SF
I in
tera
ctio
n: im
plic
atio
ns fo
r ch
aper
one
mec
hani
sm,
spec
ies-
spec
ific
inte
ract
ions
, an
d ep
igen
etic
s. B
MC
S
truc
t.Bio
l. 20
06.
v6 p
26
Age
z, M
., et
al.
Str
uctu
re o
f the
his
tone
cha
pero
ne a
sfl
bo
und
to th
e hi
ston
e h3
C-t
erm
inal
hel
ix a
nd fu
nctio
nal
insi
ghts
. S
truc
ture
200
7. v
15 p
19
l
Mou
sson
, F
., et
al.
Str
uctu
ral b
asis
for
the
inte
ract
ion
of
As
fl w
ith h
isto
ne H
3 a
nd it
s fu
nctio
nal
impl
icat
ions
. P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
5. v
102
p597
5
Nat
sum
e, R
., e
t al.
Str
uctu
re a
nd fu
nctio
n of
the
hist
one
chap
eron
e C
IAIA
SF
I co
mpl
exed
with
his
tone
s H
3 a
nd
H4.
Nat
ure
2007
. v4
46 p
338
Nat
sum
e, R
., et
al.
Str
uctu
re a
nd fu
nctio
n of
the
hist
one
chap
eron
e C
IAIA
SF
I co
mpl
exed
with
his
tone
s H
3 a
nd
H4.
Nat
ure
2007
. v4
46 p
338
2 13
2
1 CC
7
1 FE
S
1 SB
6
1 FD
8
2GG
P
1 CC
8
1 S28
1 T7S
1 l6Z
Asf
l a -
HlR
A c
ornp
lex
Atx
l
Atx
l
Atx
l
Atx
l -
Cu'
Atx
l - C
u' -
Ccc
2a
com
plex
Atx
l -
H~
''
Avr
Pph
F O
RF
2
BA
G1
(B
AG
dom
ain)
hist
one
chap
eron
e
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
prot
ein
secr
etio
n
apop
tosi
s, c
o-
chap
eron
e to
hs
p70
apop
tosi
s, c
o-
chap
eron
e to
hs
p70
X-r
ay
X-r
ay
NM
R
NM
R
NM
R,
NM
R
X-r
ay
X-r
ay
X-r
ay
NM
R
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Syn
echo
cyst
is
sp.
PC
C 6
803
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Pse
udom
onas
sy
ringa
e pv
. ph
aseo
licol
a
Cae
norh
abdi
tis
eleg
ans
Mus
mus
culu
s
Tan
g, Y
., et
al.
Str
uctu
re o
f a h
uman
AS
Fla
-HIR
A
com
plex
and
insi
ghts
into
spe
cific
ity o
f hi
ston
e ch
aper
one
com
plex
ass
embl
y. N
at.S
truc
t.Mol
.Bio
l. 20
06.
vl3
p9
2l
Ros
enzw
eig,
A.C
., et
al.
Cry
stal
str
uctu
re o
f th
e A
txl
met
allo
chap
eron
e pr
otei
n at
1.0
2 A
res
olut
ion.
Str
uctu
re
Fol
d.D
es.
1999
. v7
p60
5
Arn
esan
o, F
., et
al.
Sol
utio
n st
ruct
ure
of t
he C
u(l
) an
d a
po
form
s of
th
e y
east
met
allo
chap
eron
e, A
txl.
B
ioch
emis
try
2001
. v40
p15
28
Ban
ci,
L., e
t al
. S
olut
ion
Str
uctu
res
of a
Cya
noba
cter
ial
Met
allo
chap
eron
e: I
nsig
ht in
to a
n A
typi
cal
Cop
per-
B
indi
ng M
otif.
J.B
iol.C
hem
. 20
04.
v279
p27
502
Arn
esan
o, F
., e
t al.
Sol
utio
n st
ruct
ure
of t
he
Cu
(l)
and
ap
o fo
rms
of t
he y
east
met
allo
chap
eron
e, A
txl.
B
ioch
emis
try
2001
. v4
0 p1
528
Ban
ci,
L.,
et a
l. T
he
Atx
l-C
cc2
com
plex
is a
met
al-
med
iate
d pr
otei
n-pr
otei
n in
tera
ctio
n. N
at.C
hem
.Bio
l. 20
06.
v2 p
367
Ros
enzw
eig,
A.C
., et
al.
Cry
stal
str
uctu
re o
f th
e A
txl
met
allo
chap
eron
e pr
otei
n at
1.0
2 A
res
olut
ion.
Str
uctu
re
Fol
d.D
es.
1999
. v7
p60
5
Sin
ger,
A.U
., et
al.
Cry
stal
Str
uctu
res
of t
he T
ype
Ill
Eff
ecto
r P
rote
in A
vrP
phF
and
Its
Cha
pero
ne R
evea
l R
esid
ues
Req
uire
d fo
r P
lant
Pat
hoge
nesi
s. S
truc
ture
20
04.
v12
p16
69
Sym
ersk
y, J
., et
al.
Str
uctu
ral G
enom
ics
of
Cae
norh
abdi
tis e
lega
ns:
Str
uctu
re o
f the
BA
G d
omai
n.
Act
a C
ryst
allo
gr.,
Sec
t.D 2
004.
v60
p16
06
Bri
knar
ova,
K.,
et a
l. S
truc
tura
l ana
lysi
s of
BA
G1
coch
aper
one
and
its in
tera
ctio
ns w
ith H
sc70
hea
t sh
ock
prot
ein.
Nat
.Str
uct.B
io1.
200
1. v
8 p3
49
1 M62
2D9D
1 wxv
1 UK
5
1 UG
O
20
S7
1 P5U
1 Z9S
1 P5V
BA
G4I
SO
DD
(B
AG
do
mai
n)
BA
G-f
amily
mol
ecul
ar
chap
eron
e re
gula
tor-
5 (B
AG
dom
ain)
Bcl
-2 b
indi
ng
atha
noge
ne-I
(u
biqu
itin
dom
ain)
Bcl
2-as
soci
ated
at
hano
gene
3 (
BA
G
dom
ain)
Bcl
2-as
soci
ated
at
hano
gene
5 (
BA
G
dom
ain)
Caf
1 M
Ca
flM
- C
afl
- C
afl
co
mpl
ex
Ca
flM
- C
afl
- C
afl
co
mpl
ex
Ca
fl M
- C
aflc
ompl
ex
apop
tosi
s
n /a
n/a
apop
tosi
s
apop
tosi
s
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
NM
R
NM
R
NM
R
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Ho
mo
sap
iens
Ho
mo
sap
iens
Hom
o sa
pien
s
Mus
mus
culu
s
Mus
mus
culu
s
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Brik
naro
va, K
., et
al.
BA
G4l
SO
DD
pro
tein
con
tain
s a
shor
t B
AG
dom
ain.
J.B
iol.C
hem
. 20
02.
v277
p31
172
Hat
ta,
R.,
et a
l. S
olut
ion
stru
ctur
e of
the
BA
G d
omai
n (2
75-3
50)
of B
AG
-fam
ily m
olec
ular
cha
pero
ne r
egul
ator
- 5.
To
be p
ublis
hed.
Nira
ula,
T.N
., et
al.
Sol
utio
n st
ruct
ure
of th
e ub
iqui
tin
dom
ain
of B
CL-
2 bi
ndin
g at
hano
gene
-I.
To
be
publ
ishe
d.
Hat
ta, R
., et
al.
Sol
utio
n st
ruct
ure
of th
e M
urin
e B
AG
do
mai
n of
Bcl
2-as
soci
ated
ath
anog
ene
3. T
o b
e
publ
ishe
d.
End
oh,
H.,
et a
l. S
olut
ion
stru
ctur
e of
the
firs
t M
urin
e B
AG
dom
ain
of B
cl2-
asso
ciat
ed a
than
ogen
e 5.
To
be
pu
blis
hed.
Zav
ialo
v, A
.Z.,
Kni
ght,
S.D
. A
nov
el s
elf-
capp
ing
mec
hani
sm c
ontr
ols
aggr
egat
ion
of p
erip
lasm
ic
chap
eron
e C
afl
M.
Mol
.Mic
robi
ol.
2007
. v6
4 p
l53
Zav
ialo
v, A
.V.,
et a
l. S
truc
ture
and
Bio
gene
sis
of th
e C
apsu
lar
F1
Ant
igen
fro
m Y
ersi
nia
pest
is.
Pre
serv
ed
Fol
ding
Ene
rgy
Driv
es F
iber
For
mat
ion.
Cel
l 200
3. v
113
p587
Zav
ialo
v, A
.V.,
et a
l. R
esol
ving
the
ener
gy p
arad
ox o
f ch
aper
one/
ushe
r-m
edia
ted
fibre
ass
embl
y. B
i0ch
em.J
. 20
05.
v389
p68
5
Zav
ialo
v, A
.V.,
et a
l. S
truc
ture
and
Bio
gene
sis
of th
e C
apsu
lar
F1
Ant
igen
fro
m Y
ersi
nia
pest
is.
Pre
serv
ed
Fol
ding
Ene
rgy
Driv
es F
iber
For
mat
ion.
Cel
l 200
3. v
113
p587
1 JH
N
1 HH
N
1 J6Q
1 LM
O
1 SR
3
1 GM
L
1 GN
1
1 XO
U
1 K3E
1WN
R
Cal
nexi
n (lu
men
al
dom
ain)
Cal
retic
ulin
(P-d
omai
n)
Ccm
E
Ccm
E
Ccm
E
CC
T (
gam
ma
apic
al
dom
ain)
CC
T (
gam
ma
apic
al
dom
ain)
Ces
A -
Esp
A c
ompl
ex
Ces
T
Cha
pero
nin
10
prot
ein
fold
ing
prot
ein
fold
ing
hem
e ch
aper
one
hem
e ch
aper
one
hem
e ch
aper
one
prot
ein
fold
ing
prot
ein
fold
ing
type
Ill
prot
ein
secr
etio
n
type
Ill
prot
ein
secr
etio
n
prot
ein
fold
ing,
G
roE
S-li
ke
X-r
ay
NM
R
NM
R
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Can
is fa
mili
aris
Rat
tus
norv
egic
us
She
wan
ella
pu
tref
acie
ns
She
wan
ella
pu
tref
acie
ns
Esc
heric
hia
coli
Mu
s m
uscu
lus
Mu
s m
uscu
lus
Esc
heric
hia
coli
Esc
heric
hia
coli
The
rmus
th
erm
ophi
lus
Sch
rag,
J.D
., et
al.
The
Str
uctu
re o
f cal
nexi
n, a
n E
R
chap
eron
e in
volv
ed in
qua
lity
cont
rol o
f pro
tein
fold
ing.
M
oLC
ell 2
001.
v8
p633
Ellg
aard
, L.,
et a
l. N
MR
str
uctu
re o
f the
cal
retic
ulin
P-
dom
ain.
Pro
c.N
atl.A
cad.
Sci
.US
A 2
001.
v98
p31
33
Arn
esan
o, F
., et
al.
Sol
utio
n st
ruct
ure
and
char
acte
rizat
ion
of th
e he
me
chap
eron
e C
cmE
. B
ioch
emis
try
2002
. v41
p13
587
Arn
esan
o, F
., et
al.
Sol
utio
n st
ruct
ure
and
char
acte
rizat
ion
of th
e he
me
chap
eron
e C
cmE
. B
ioch
emis
try
2002
. v4
1 p1
3587
Eng
gist
, E.,
et a
l. N
MR
Str
uctu
re o
f the
Hem
e C
hape
rone
Ccm
e R
evea
ls a
Nov
el F
unct
iona
l Mot
if.
Str
uctu
re 2
002.
v10
pl5
5l
Pap
penb
erge
r, G
., et
al.
Cry
stal
str
uctu
re o
f the
C
CT
gam
ma
apic
al d
omai
n: im
plic
atio
ns fo
r su
bstr
ate
bind
ing
to th
e eu
kary
otic
cyt
osol
ic c
hape
roni
n.
J.M
ol.B
iol.
2002
. v3
18 p
1367
Pap
penb
erge
r, G
., et
al.
Cry
stal
str
uctu
re o
f the
C
CT
gam
ma
apic
al d
omai
n: im
plic
atio
ns fo
r su
bstr
ate
bind
ing
to th
e eu
kary
otic
cyt
osol
ic c
hape
roni
n.
J.M
ol.B
iol.
2002
. v31
8 p1
367
Yip
, C
.K.,
Fin
lay,
B.B
., S
tryn
adka
, N
.C.J
. S
truc
tura
l ch
arac
teriz
atio
n of
a ty
pe I
ll se
cret
ion
syst
em fi
lam
ent
prot
ein
in c
ompl
ex w
ith it
s ch
aper
one.
N
at.S
truc
t.Mol
.Bio
l. 200
5. v
12 p
75
Luo,
Y.,
et a
l. S
truc
tura
l and
bio
chem
ical
cha
ract
eriz
atio
n of
the
type
Ill
secr
etio
n ch
aper
ones
Ces
T a
nd S
igE
. N
at.S
truc
t.Bio
l. 20
01. v
8 p1
031
Num
oto,
N.,
et a
l. C
ryst
al s
truc
ture
of t
he C
o-ch
aper
onin
C
pnlO
from
The
rmus
ther
mop
hilu
s H
B8.
Pro
tein
s 20
05.
v58
p498
1WE
3
1WF
4
ISJP
1 HX
5
1 LE
P
1 P3H
1 P82
1 P83
1 IO
K
Cha
pero
nin
60
- C
hape
roni
n 10
- (A
DP
)7 c
ompl
ex
prot
ein
fold
ing,
G
roE
L-G
roE
S
like
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
X-r
ay
The
rmus
th
erm
ophi
lus
Shi
mam
ura,
T.,
et a
l. C
ryst
al s
truc
ture
of t
he n
ativ
e ch
aper
onin
com
plex
fro
m T
herm
us t
herm
ophi
lus
reve
aled
une
xpec
ted
asym
met
ry a
t the
cis
-cav
ity.
Str
uctu
re 2
004.
v12
p14
71
Shi
mam
ura,
T.,
et a
l. C
ryst
al s
truc
ture
of t
he n
ativ
e ch
aper
onin
com
plex
fro
m T
herm
us t
herm
ophi
lus
reve
aled
une
xpec
ted
asym
met
ry a
t th
e ci
s-ca
vity
. S
truc
ture
200
4. v
12 p
1471
Cha
pero
nin
60 -
Cha
pero
nin
10 -
(AD
P)7
com
plex
prot
ein
fold
ing,
G
roE
L-G
roE
S
like
The
rmus
th
erm
ophi
lus
Cha
pero
nin
60.2
pr
otei
n fo
ldin
g,
Gro
EL-
like
Myc
obac
teriu
m
tube
rcul
osis
Q
amra
, R
., an
d M
ande
, S
.C.
Cry
stal
Str
uctu
re o
f the
65
- K
iloda
lton
Hea
t Sho
ck P
rote
in,
Cha
pero
nin
60.2
, of
M
ycob
acte
rium
tub
ercu
losi
s. J
.Bac
terio
l. 20
04.
v186
p8
105
Tan
eja,
B.,
and
Man
de,
S.C
. Thr
ee-d
imen
sion
al
Str
uctu
re o
f M
ycob
acte
rium
tube
rcul
osis
Cha
pero
nin-
10
Rev
eals
a P
artia
lly S
tabl
e C
onfo
rmat
ion
for
its M
obile
Lo
op. C
urr.
Sci
. 20
01. v
81 p
87
Myc
obac
teriu
m
tube
rcul
osis
pr
otei
n fo
ldin
g,
Gro
ES
-like
prot
ein
fold
ing,
G
roE
S-li
ke
Myc
obac
teriu
m
lepr
ae
Man
de,
S.C
., et
al.
Str
uctu
re o
f the
hea
t sho
ck p
rote
in
chap
eron
in-1
0 of
Myc
obac
teriu
m le
prae
. S
cien
ce 1
996.
v2
71 p
203
Rob
erts
, M
.M.,
et a
l. M
ycob
acte
rium
tube
rcul
osis
ch
aper
onin
10
hept
amer
s se
lf-as
soci
ate
thro
ugh
thei
r bi
olog
ical
ly a
ctiv
e lo
ops.
J.B
acte
riol.
2003
. v1
85 p
4172
prot
ein
fold
ing,
G
roE
S-li
ke
Myc
obac
teriu
m
tube
rcul
osis
Cha
pero
nin-
10 (
N-
term
inal
dom
ain)
pr
otei
n fo
ldin
g,
Gro
ES
-like
sy
nthe
tic
Ciu
tti, A
,, et
al.
Sol
utio
n S
truc
ture
of
1-25
frag
men
t of
C
pn
lO fr
om M
ycob
acte
rium
Tub
ercu
losi
s. T
o be
pu
blis
hed.
prot
ein
fold
ing,
G
roE
S-li
ke
synt
hetic
C
iutti
, A.,
et a
l. S
olut
ion
Str
uctu
re o
f 1-
25 fr
agm
ent o
f C
pn
lO fr
om M
ycob
acte
rium
Tub
ercu
losi
s. T
o be
pu
blis
hed.
Cha
pero
nin-
1 0
(N-
term
inal
dom
ain)
prot
ein
fold
ing,
G
roE
L-lik
e P
arac
occu
s de
nitr
ifica
ns
Fuk
ami,
T.A
., et
al.
Cry
stal
str
uctu
re o
f cha
pero
nin-
60
from
Par
acoc
cus
deni
trifi
cans
. J.
Mol
.Bio
l. 20
01.
v312
p5
01
CH
IP (
C-t
erm
inal
do
mai
n)
co-c
hape
rone
X
-ray
to
hsp
70
2.5
D
an
iore
rio
X
u, Z
., et
al.
Str
uctu
re a
nd I
nter
actio
ns o
f the
Hel
ical
an
d
U-B
ox D
omai
ns o
f C
HIP
, th
e C
Ter
min
us o
f H
SP
7O
Inte
ract
ing
Pro
tein
. B
ioch
emis
try
2006
. v4
5 p4
749
1.8
Sch
izos
acch
aro
Pad
man
abha
n, B
., an
d Y
okoy
ama,
S.
Cry
stal
str
uctu
re
myc
es p
ombe
o
f H
isto
ne c
hape
rone
cia
l. T
o b
e p
ublis
hed.
C
ia I
hist
one
X-r
ay
chap
eron
e
nucl
eoso
me
X-r
ay
asse
mbl
y 3.
0 S
acch
arom
yces
P
adm
anab
han,
B.,
et a
l. S
truc
tura
l Sim
ilarit
y be
twee
n ce
revi
siae
H
isto
ne C
hape
rone
Cia
1 p
lAsf
l p a
nd D
NA
-Bin
ding
P
rote
in N
F-{
kapp
a}B
. J.
Bio
chem
.(T
okyo
) 20
05.
v138
p8
21
2.75
H
omo
sapi
ens
Gho
sh,
P.,
et a
l. T
he s
truc
ture
of
an in
term
edia
te i
n cl
ass
II
MH
C m
atur
atio
n: C
LIP
bou
nd to
HLA
-DR
3. N
atur
e 19
95. v
378
p457
X-r
ay
CLI
P -
HLA
-DR
3 co
mpl
ex
imm
une
resp
onse
, an
tigen
pr
oces
sing
an
d pr
esen
tatio
n
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
Clp
A
1.8
Esc
heric
hia
coli
Guo
, F
., et
al.
Cry
stal
str
uctu
re o
f Clp
A,
an H
SP
IOO
ch
aper
one
and
regu
lato
r o
f C
lpA
P p
rote
ase.
J.
Bio
l.Che
m. 2
002.
v27
7 p4
6743
1 K6K
1 R6B
1 KS
F
1 R6C
X-r
ay
Clp
A
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
X-r
ay
2.25
E
sche
richi
a co
li X
ia,
D.,
et a
l. C
ryst
allo
grap
hic
inve
stig
atio
n of
pep
tide
bind
ing
site
s in
the
N-d
omai
n o
f th
e C
lpA
cha
pero
ne.
J.S
truc
t.Bio
l. 20
04. v
146
p166
Clp
A -
AD
P
prot
ein
X-r
ay
2.6
Esc
heric
hia
coli
Guo
, F
., et
al.
Cry
stal
str
uctu
re o
f Clp
A,
an H
SP
IOO
ch
aper
one
and
regu
lato
r of
Clp
AP
pro
teas
e.
J.B
iol.C
hem
. 20
02.
v277
p46
743
degr
adat
ion
(in
co
mpl
ex w
ith
C~
PP
)
prot
ein
degr
adat
ion
(in
Clp
A (
N-t
erm
inal
do
mai
n)
2.15
E
sche
richi
a co
li X
ia,
D.,
et a
l. C
ryst
allo
grap
hic
inve
stig
atio
n o
f pe
ptid
e bi
ndin
g si
tes
in th
e N
-dom
ain
of
the
Clp
A c
hape
rone
. J.
Str
uct.B
iol.
2004
. v1
46 p
166
X-r
ay
com
plex
with
C
~P
P)
1 LZ
W
Clp
A (
N-t
erm
inal
do
mai
n) -
Clp
S
com
plex
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Esc
heric
hia
coli
Zet
h, K
., et
al.
Str
uctu
ral a
naly
sis
of th
e ad
apto
r pr
otei
n C
lpS
in c
ompl
ex w
ith t
he N
-ter
min
al d
omai
n of
Clp
A.
Nat
.Str
uct.B
iol.
2002
. v9
p906
1 MB
U
Clp
A (
N-t
erm
inal
do
mai
n) -
Clp
S
com
plex
prot
ein
degr
adat
ion
(in
com
plex
with
C
IPP
)
Esc
heri
chia
co
li G
uo,
F.,
et a
l. C
ryst
al S
truc
ture
of t
he H
eter
odim
eric
C
ompl
ex o
f th
e A
dapt
or,
Clp
S, w
ith t
he N
-dom
ain
of th
e A
AA
+ C
hape
rone
, C
lpA
. J.B
iol.C
hem
. 20
02. v
277
p467
53
1 MB
V
Clp
A (
N-t
erm
inal
do
mai
n) -
Clp
S
com
plex
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
Esc
heric
hia
coli
Guo
, F
., et
al.
Cry
stal
Str
uctu
re o
f the
Het
erod
imer
ic
Com
plex
of t
he A
dapt
or,
Clp
S, w
ith t
he N
-dom
ain
of
AM
+ C
hape
rone
Clp
A.
J.B
iol.C
hem
. 20
02. v
277
p467
53
Guo
, F.
, et
al. C
ryst
al S
truc
ture
of t
he H
eter
odim
eric
C
ompl
ex o
f th
e A
dapt
or,
Clp
S, w
ith t
he N
-dom
ain
of th
e A
AA
+ C
hape
rone
, C
lpA
. J.B
iol.C
hem
. 20
02. v
277
p467
53
1 MB
X
Clp
A (
N-t
erm
inal
do
mai
n) -
Clp
S
com
plex
Esc
heric
hia
coli
1 MG
9 C
lpA
(N
-ter
min
al
dom
ain)
- C
lpS
co
mpl
ex
prot
ein
degr
adat
ion
(in
com
plex
with
C
IPP
)
Esc
heric
hia
coli
Zet
h, K
., et
al.
Str
uctu
ral a
naly
sis
of th
e ad
apto
r pr
otei
n C
lpS
in c
ompl
ex w
ith t
he N
-ter
min
al d
omai
n of
Clp
A.
Nat
.Str
uct.B
iol.
2002
. v9
p906
1 R6
0
Clp
A (
N-t
erm
inal
do
mai
n) -
Clp
S
com
plex
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
Esc
heric
hia
coli
Xia
, D
., et
al.
Cry
stal
logr
aphi
c in
vest
igat
ion
of p
eptid
e bi
ndin
g si
tes
in th
e N
-dom
ain
of t
he C
lpA
cha
pero
ne.
J.S
truc
t.Bio
l. 20
04. v
146
p166
1 R6Q
C
lpA
(N
-ter
min
al
dom
ain)
- C
lpS
co
mpl
ex
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
Esc
heric
hia
coli
Xia
, D
., et
al.
Cry
stal
logr
aphi
c in
vest
igat
ion
of p
eptid
e bi
ndin
g si
tes
in th
e N
-dom
ain
of t
he C
lpA
cha
pero
ne.
J.S
truc
t.Bio
l. 20
04. v
146
p166
IQV
R
Clp
B
reso
lubi
lizat
ion
of p
rote
in
aggr
egat
es
The
rmus
th
erm
ophi
lus
Lee,
S.,
et a
l. T
he S
truc
ture
of
Clp
B: A
Mol
ecul
ar
Cha
pero
ne th
at R
escu
es P
rote
ins
from
an
Agg
rega
ted
Sta
te. C
ell 2
003.
v11
5 p2
29
1 JB
K
1 KH
Y
2P65
1 UM
8
1 OV
X
1 YG
O
1 QU
P
2CR
L
Clp
B (
NB
DI)
Clp
B (
N-t
erm
inal
do
mai
n)
Clp
Bl
(NB
DI
dom
ain)
Clp
X
Clp
X (
zinc
bin
ding
do
mai
n)
Cop
P
copp
er c
hape
rone
for
supe
roxi
de d
ism
utas
e
copp
er c
hape
rone
for
supe
roxi
de d
ism
utas
e (H
MA
dom
ain)
reso
lubi
lizat
ion
of p
rote
in
aggr
egat
es
reso
lubi
lizat
ion
of p
rote
in
aggr
egat
es
reso
lubi
lizat
ion
of p
rote
in
aggr
egat
es
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
prot
ein
degr
adat
ion
(in
com
plex
with
C
~P
P)
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
X-r
ay
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
X-r
ay
NM
R
Esc
heric
hia
coli
Esc
heric
hia
coli
Pla
smod
ium
fa
lcip
arum
Hel
icob
acte
r py
lori
2669
5
Esc
heric
hia
coli,
E
sche
richi
a co
li 0
6, E
sche
richi
a co
li 0
15
7:H
7,
and
Shi
gella
fle
xner
i
Hel
icob
acte
r py
lori
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Li, J
., S
ha,
B. C
ryst
al s
truc
ture
of
E. c
oli H
splO
O C
lpB
nu
cleo
tide-
bind
ing
dom
ain
1 (N
BD
I) a
nd m
echa
nist
ic
stud
ies
on C
lpB
AT
Pas
e ac
tivity
. J.
Mol
.Bio
l. 20
02.
v318
p
ll2
7
Jing
zhi,
L.,
Bin
gdon
g, S
. The
Cry
stal
Str
uctu
re o
f E
. col
i H
splO
O C
lpB
N T
erm
inal
Dom
ain,
Im
plic
atio
n to
Pep
tide
Bin
ding
Fun
ctio
n of
Clp
B. T
o be
pub
lishe
d.
Wer
nim
ont,
A.K
., et
al.
Cry
stal
Str
uctu
re o
f the
firs
t nu
cleo
tide
bind
ing
dom
ain
of c
hape
rone
Clp
B1,
put
ativ
e,
(Pv0
8958
0) f
rom
Pla
smod
ium
Viv
ax.
To b
e pu
blis
hed.
Kim
, D.Y
., K
im, K
.K. C
ryst
al S
truc
ture
of
Clp
X M
olec
ular
C
hape
rone
from
Hel
icob
acte
r py
lori.
J.B
iol.C
hem
. 20
03.
v278
p50
664
Don
alds
on,
L.W
., W
ojty
ra,
U.,
Hou
ry, W
.A.
Sol
utio
n st
ruct
ure
of th
e di
mer
ic z
inc
bind
ing
dom
ain
of th
e ch
aper
one
Clp
X. J
.Bio
l.Che
m.
2003
. v27
8 p4
8991
Lee,
B.J
., P
ark,
S.J
. S
olut
ion
stru
ctur
e of
apo
-Cop
P f
rom
H
elic
obac
ter
pylo
ri. T
o be
pub
lishe
d.
Lam
b, A
.L.,
et a
l. C
ryst
al s
truc
ture
of t
he c
oppe
r ch
aper
one
for
supe
roxi
de d
ism
utas
e. N
at.S
truc
t.Bio
l 19
99. v
6 p7
24
Nag
ashi
ma,
T.,
et a
l. T
he a
po fo
rm o
f H
MA
dom
ain
of
copp
er c
hape
rone
for
sup
erox
ide
dism
utas
e. T
o be
pu
blis
hed.
1 JK
9
1 CP
Z
1 P8G
2HU
9
1 U97
1 Z2G
1 U96
1 GD
7
2NZ
H
2N
Z0
copp
er c
hape
rone
for
su
pero
xide
dis
mut
ase
(yC
CB
) - s
uper
oxid
e di
smut
ase
(yS
OD
1)
com
plex
co
pz
co
pz
Co
pZ
(N-t
erm
inal
do
mai
n)
Co
xl 7
Co
xl 7
Co
xl 7
- C
u'
Csa
A
Csa
A
Csa
A
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
prot
ein
secr
etio
n
prot
ein
secr
etio
n
prot
ein
secr
etio
n
X-r
ay
NM
R
NM
R
X-r
ay
NM
R
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
cere
visi
ae
Ent
eroc
occu
s hi
rae
Bac
illus
sub
tilis
Arc
haeo
glob
us
fulg
idus
Sac
char
omyc
es
ce re
visi
a e
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
The
rmus
th
erm
ophi
lus
Bac
illus
sub
tilis
Bac
illus
sub
tilis
Lam
b, A
.L.,
et a
l. H
eter
odim
eric
str
uctu
re o
f su
pero
xide
di
smut
ase
in c
ompl
ex w
ith i
ts m
etal
loch
aper
one.
N
at.S
truc
t.B
iol.
2001
. v8
p751
Wim
mer
, R
., et
al.
NM
R s
truc
ture
and
met
al in
tera
ctio
ns
of t
he C
opZ
cop
per
chap
eron
e. J
.Bio
l.Che
m.
1999
. v27
4 p2
2597
Ban
ci, L
., et
al.
Sol
utio
n S
truc
ture
of A
po C
opZ
from
B
acill
us s
ubtil
is:
Fur
ther
Ana
lysi
s of
the
Cha
nges
A
ssoc
iate
d w
ith th
e P
rese
nce
of C
oppe
r. B
ioch
emis
try
2003
. v4
2 ~
13
42
2
Saz
insk
y, M
.H.,
et a
l. C
hara
cter
izat
ion
and
Str
uctu
re o
f a
Nov
el Z
n2+
an
d [2
Fe-
2S]-
Con
tain
ing
Cop
per
Cha
pero
ne
from
Arc
haeo
glob
us f
ulgi
dus.
To
be
pub
lishe
d.
Aba
jian,
C.,
et a
l. Y
east
cox
17 s
olut
ion
stru
ctur
e an
d C
oppe
r(1)
bin
ding
. J.
Bio
l.Che
m. 2
004.
v27
9 p5
3584
Arn
esan
o, F
., et
al.
Fol
ding
stu
dies
of
Cox
17 r
evea
l an
im
port
ant i
nter
play
of
cyst
eine
oxi
datio
n a
nd
cop
per
bind
ing.
Str
uctu
re 2
005.
v13
p71
3
Aba
jian,
C.,
et a
l. Y
east
cox
17 s
olut
ion
stru
ctur
e an
d C
oppe
r(1)
bin
ding
. J.
Bio
l.Che
m. 2
004.
v27
9 p5
3584
Kaw
aguc
hi,
S.,
et a
l. T
he c
ryst
al s
truc
ture
of
the
ttCsa
A
prot
ein:
an
exp
ort-
rela
ted
chap
eron
e fr
om T
herm
us
ther
mop
hilu
s. E
MB
O J
. 200
1. v
20 p
562
Sha
pova
, Y
.A.,
Pae
tzel
, M.
Cry
stal
logr
aphi
c an
alys
is o
f B
acill
us s
ubtil
is C
saA
. Act
a C
ryst
allo
gr.,S
ect.D
200
7.
v63
p478
Sha
pova
, Y
.A.,
Pae
tzel
, M.
Cry
stal
logr
aphi
c an
alys
is o
f B
acill
us s
ubtil
is C
saA
. Act
a C
ryst
allo
gr.,S
ect.D
200
7.
v63
p478
3ME
F
1XM
L
1XM
M
1 KY
9
1 IB
X
1 IB
X
1 EX
K
1 BQ
O
Csp
A
Dcp
S
Dcp
S -
m7G
DP
co
mp
lex
De
gP
(Htr
A)
DF
F4
0 (
N-t
erm
inal
do
mai
n) -
DF
F45
(N-
term
inal
dom
ain)
co
mp
lex
DF
F4
0 (N
-ter
min
al
dom
ain)
- D
FF
45 (
N-
term
inal
dom
ain)
co
mp
lex
Dn
aJ
(cys
tein
e-ric
h d
om
ain
)
Dn
aJ
(J-d
omai
n)
regu
latio
n of
tr
ansc
riptio
n
prot
ease
- ch
aper
one
apop
tosi
s
apop
tosi
s
prot
ein
fold
ing,
co
-cha
pero
ne
to D
naK
, E
.col
i hs
p40
prot
ein
fold
ing,
co
-cha
pero
ne
to D
naK
, E.c
oli
hsp4
0
NM
R
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
NM
R
NM
R
Esc
heri
chia
col
i
Hom
o sa
pien
s
Hom
o sa
pien
s
Esc
heri
chia
col
i
Hom
o sa
pien
s
Str
epto
cocc
us
sp.
and
Hom
o sa
pien
s
Esc
heri
chia
col
i
Esc
heri
chia
col
i
Fen
g, W
., et
al.
Sol
utio
n N
MR
str
uctu
re a
nd b
ackb
one
dyna
mic
s of
th
e m
ajor
col
d-sh
ock
prot
ein
(Csp
A) f
rom
E
sche
richi
a co
li: e
vide
nce
for
conf
orm
atio
nal
dyna
mic
s in
the
sing
le-s
tran
ded
RN
A-b
indi
ng s
ite.
Bio
chem
istr
y 19
98. v
37 ~
10
88
1
Che
n, N
., e
t al.
Cry
stal
Str
uctu
res
of H
uman
Dcp
S in
Li
gand
-fre
e an
d m
7GD
P-b
ound
for
ms
Sug
gest
a
Dyn
amic
Mec
hani
sm fo
r S
cave
nger
rnR
NA
Dec
appi
ng.
J.M
ol.B
iol.
2005
. v3
47 p
707
Che
n, N
., e
t al.
Cry
stal
Str
uctu
res
of H
uman
Dcp
S in
Li
gand
-fre
e a
nd
m7G
DP
-bou
nd f
orm
s S
ugge
st a
D
ynam
ic M
echa
nism
for
Sca
veng
er m
RN
A D
ecap
ping
. J.
Mol
.Bio
l. 20
05.
v347
p70
7
Kro
jer,
T.,
et a
l. C
ryst
al s
truc
ture
of
Deg
P (
Htr
A)
reve
als
a ne
w p
rote
ase-
chap
eron
e m
achi
ne.
Nat
ure
2002
. v41
6 p4
55
Zho
u, P
., et
al.
Sol
utio
n st
ruct
ure
of D
FF
40 a
nd D
FF
45
N-t
erm
inal
dom
ain
com
plex
an
d m
utua
l cha
pero
ne
activ
ity o
f D
FF
40 a
nd D
FF
45. P
roc.
Nat
l.Aca
d.S
ci.U
SA
20
01. v
98 p
6051
Zho
u, P
., et
al.
Sol
utio
n st
ruct
ure
of D
FF
40 a
nd D
FF
45
N-t
erm
inal
dom
ain
com
plex
and
mut
ual c
hape
rone
ac
tivity
of
DF
F40
and
DF
F45
. P
roc.
Nat
l.Aca
d.S
ci.U
SA
20
01.
v98
p605
1
Mar
tinez
-Yam
out,
M.,
et a
l. S
olut
ion
stru
ctur
e of
the
cy
stei
ne-r
ich
dom
ain
of th
e E
sche
richi
a co
li ch
aper
one
prot
ein
Dna
J. J
.Mol
.Bio
l. 20
00.
v300
p80
5
Hua
ng,
K.,
et a
l. T
he in
fluen
ce o
f C
-ter
min
al e
xten
sion
on
the
stru
ctur
e of
the
'J-d
omai
n' in
E. c
oli D
naJ.
Pro
tein
S
ci.
1999
. v8
p2
03
1 BQ
Z
Dna
J (J
-dom
ain)
1 XB
L D
naJ
(J-d
omai
n)
2CT
P
2CT
Q
2CTR
2C
W
2DM
X
2DN
9
2CT
T
IWJZ
1 DK
G
Dna
J ho
mol
og (
J-
dom
ain)
Dna
J ho
mol
og (
J-
dom
ain)
Dna
J ho
mol
og (
J-
dom
ain)
Dna
J ho
mol
og (
J-
dom
ain)
Dna
J ho
mol
og (
J-
dom
ain)
Dna
J ho
mol
og (T
id I,
J-do
mai
n)
Dna
J ho
mol
og (
zinc
fin
ger d
omai
n)
Dna
J-lik
e pr
otei
n (J
- do
mai
n)
Dna
K (
AT
Pas
e do
mai
n) -
Grp
E
com
plex
prot
ein
fold
ing,
co
-cha
pero
ne
to D
naK
, E.c
oli
hsp4
0
prot
ein
fold
ing,
co
-cha
pero
ne
to D
naK
, E.c
oli
hsp4
0
nla
prot
ein
fold
ing,
E
. co
li hs
p70
NM
R
NM
R
NM
R
NM
R
NM
R
NM
R
NM
R
NM
R
NM
R
NM
R
X-r
ay
nla
nla
nla
nla
nla
nla
nla
nla
nla
nla
2.8
Esc
heric
hia
coli
Esc
heric
hia
coli
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Mus
mus
culu
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Mus
mus
culu
s
Esc
heric
hia
coli
Hua
ng, K
., et
al.
The
influ
ence
of
C-t
erm
inal
ext
ensi
on
on th
e st
ruct
ure
of th
e 'J
-dom
ain'
in E
. col
i Dna
J. P
rote
in
Sci
. 19
99. v
8 p2
03
Pel
lecc
hia,
M.,
et a
l. N
MR
str
uctu
re o
f the
J-d
omai
n an
d th
e G
lyIP
he-r
ich
regi
on o
f the
Esc
heric
hia
coli
Dna
J ch
aper
one.
J.M
ol.B
iol.
1996
. v26
0 p2
36
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of J
-dom
ain
from
hu
man
Dna
J su
bfam
ily B
men
ber
12. T
o be
pub
lishe
d.
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of J
-dom
ain
from
hu
man
Dna
J su
bfam
ily C
men
ber
12. T
o be
pub
lishe
d.
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of J
-dom
ain
from
hu
man
Dna
J su
bfam
ily B
men
ber 9
. To
be p
ublis
hed.
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of J
-dom
ain
from
m
ouse
Dna
J su
bfam
ily C
men
ber
5. T
o be
pub
lishe
d.
Ohn
ishi
, S.,
et a
l. S
olut
ion
stru
ctur
e of
the
J do
mai
n of
D
naJ
hom
olog
sub
fam
ily B
mem
ber 8
. To
be
publ
ishe
d.
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of J
-dom
ain
from
th
e D
naJ
hom
olog
, hum
an T
idl
prot
ein.
To
be p
ublis
hed.
Kob
ayas
hi, N
., et
al.
Sol
utio
n st
ruct
ure
of z
inc
finge
r do
mai
n fr
om h
uman
Dna
J su
bfam
ily A
men
ber 3
. To
be
publ
ishe
d.
Kob
ayas
hi, N
., et
al.
Sol
uiot
n st
ruct
ure
of J
-dom
ain
of
mou
se D
naJ
like
prot
ein.
To
be p
ublis
hed.
Har
rison
, C.J
., et
al.
Cry
stal
str
uctu
re o
f the
nuc
leot
ide
exch
ange
fact
or G
rpE
bou
nd to
the
AT
Pas
e do
mai
n of
th
e m
olec
ular
cha
pero
ne D
naK
. Sci
ence
199
7. v
276
p431
1 BP
R
Dna
K (
subs
trat
e bi
ndin
g do
mai
n)
prot
ein
fold
ing,
E
.col
i hsp
70
NM
R
Esc
heric
hia
coli
Wan
g, H
., et
al.
NM
R s
olut
ion
stru
ctur
e of
the
21
kDa
chap
eron
e pr
otei
n D
naK
sub
stra
te b
indi
ng d
omai
n: a
pr
evie
w o
f cha
pero
ne-p
rote
in in
tera
ctio
n. B
ioch
emis
try
1998
. v37
p79
29
Dna
K (
subs
trat
e bi
ndin
g do
mai
n)
prot
ein
fold
ing,
E
. col
i hsp
70
NM
R
NM
R
Esc
heric
hia
coli
Pel
lecc
hia,
M.,
et a
l. S
truc
tura
l ins
ight
s in
to s
ubst
rate
bi
ndin
g by
the
mol
ecul
ar c
hape
rone
Dna
K.
Nat
.Str
uct.B
iol.
2000
. v7
p29
8
Dna
K (
subs
trat
e bi
ndin
g do
mai
n)
prot
ein
fold
ing,
E
. co
li hs
p70
Esc
heric
hia
coli
Wan
g, H
., et
al.
NM
R s
olut
ion
stru
ctur
e of
the
21
kDa
chap
eron
e pr
otei
n D
naK
sub
stra
te b
indi
ng d
omai
n: a
pr
evie
w o
f cha
pero
ne-p
rote
in in
tera
ctio
n. B
ioch
emis
try
1998
. v37
p79
29
1 D
KX
1 DK
Y
1 DK
Z
1 Q
5L
Dna
K (
subs
trat
e bi
ndin
g do
mai
n) -
pept
ide
com
plex
prot
ein
fold
ing,
E
. co
li hs
p70
X-r
ay
X-r
ay
X-r
ay
NM
R
Esc
heric
hia
coli
Zhu
, X
., et
al.
Str
uctu
ral a
naly
sis
of s
ubst
rate
bin
ding
by
the
mol
ecul
ar c
hape
rone
Dna
K. S
cien
ce 1
996.
v27
2 p
l6O
6
Dna
K (
subs
trat
e bi
ndin
g do
mai
n) -
pept
ide
com
plex
prot
ein
fold
ing,
E
. col
i hsp
70
Esc
heric
hia
coli
Zhu
, X
., et
al.
Str
uctu
ral a
naly
sis
of s
ubst
rate
bin
ding
by
the
mol
ecul
ar c
hape
rone
Dna
K. S
cien
ce 1
996.
v27
2 pl
6O6
Dna
K (
subs
trat
e bi
ndin
g do
mai
n) -
pe
ptid
e co
mpl
ex
prot
ein
fold
ing,
E
. col
i hsp
70
Esc
heric
hia
coli
Zhu
, X.,
et a
l. S
truc
tura
l ana
lysi
s of
sub
stra
te b
indi
ng b
y th
e m
olec
ular
cha
pero
ne D
naK
. Sci
ence
199
6. v
272
p160
6
Dna
K (
subs
trat
e bi
ndin
g do
mai
n) -
pept
ide
com
plex
prot
ein
fold
ing,
E. c
oli h
sp70
E
sche
richi
a co
li S
teve
ns, S
.Y.,
et a
l. T
he s
olut
ion
stru
ctur
e of
the
ba
cter
ial H
SP
70 c
hape
rone
pro
tein
dom
ain
Dna
K(3
93-
507)
in c
ompl
ex w
ith th
e pe
ptid
e N
RLL
LTG
. Pro
tein
Sci
. 20
03.
v12
p258
8
Osi
piuk
, J.,
et a
l. X
-ray
cry
stal
str
uctu
re o
f J-d
omai
n of
dn
j-12
from
Cae
norh
abdi
tis e
lega
ns.
To
be p
ublis
hed.
X
-ray
X-r
ay
Cae
norh
abdi
tis
eleg
ans
prot
ein
fold
ing
Dsb
G
Esc
heric
hia
coli
Her
as, B
., et
al.
Cry
stal
str
uctu
res
of t
he D
sbG
dis
ulfid
e is
omer
ase
reve
al a
n un
stab
le d
isul
fide.
P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
4. v
lOl
p887
6
Dsb
G
prot
ein
fold
ing
X-r
ay
Esc
heric
hia
coli
Her
as, B
., et
al.
Cry
stal
str
uctu
res
of t
he D
sbG
dis
ulfid
e is
omer
ase
reve
al a
n un
stab
le d
isul
fide.
P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
4. v
l01
p88
76
2HO
G
2HO
H
2HO
I
1 EX
M
1 G7D
1 G7E
2ALB
2BS
G
Dsb
G (
mut
ant)
pr
otei
n fo
ldin
g X
-ray
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
NM
R
Cry
o-
EM
X-r
ay
Esc
heric
hia
coli
Hin
iker
, A.,
et a
l. S
hort
-circ
uitin
g di
verg
ent e
volu
tion:
la
bora
tory
evo
lutio
n of
one
dis
ulfid
e Is
omer
ase
to
rese
mbl
e an
othe
r. T
o be
pub
lishe
d.
Hin
iker
, A.,
et a
l. S
hort
-circ
uitin
g di
verg
ent
evol
utio
n:
labo
rato
ry e
volu
tion
of o
ne d
isul
fide
isom
eras
e to
re
sem
ble
anot
her.
To
be p
ublis
hed.
prot
ein
fold
ing
Esc
heric
hia
coli
Dsb
G (
mut
ant)
Dsb
G (
mut
ant)
pr
otei
n fo
ldin
g E
sche
richi
a co
li H
inik
er, A
., et
al.
Sho
rt-c
ircui
ting
dive
rgen
t ev
olut
ion:
la
bora
tory
evo
lutio
n of
one
dis
ulfid
e is
omer
ase
to
rese
mbl
e an
othe
r. T
o be
pub
lishe
d.
Hilg
enfe
ld,
R.,
et a
l. In
sigh
ts in
to th
e G
TP
ase
Mec
hani
sm o
f E
F-T
U fr
om S
truc
tura
l Stu
dies
. The
E
long
atio
n fa
ctor
Tu
(EF
-Tu)
- G
PP
NH
P
tran
slat
ion,
pr
otei
n fo
dlin
g?
The
rmus
th
erm
ophi
lus
com
plex
. R
ibos
ome:
Str
uctu
re,
Fun
ctio
n, A
ntib
iotic
s, a
nd C
ellu
lar
Inte
ract
ions
200
0. v
28 p
347
Raf
f us
norv
egic
us
Liep
insh
, E
., et
al.
Thi
ored
oxin
fold
as
hom
odim
eriz
atio
n m
odul
e in
the
puta
tive
chap
eron
e E
Rp2
9: N
MR
st
ruct
ures
of t
he d
omai
ns a
nd e
xper
imen
tal
mod
el o
f the
51
kD
a di
mer
. Str
uctu
re 2
001.
v9
p457
Raf
fus
norv
egic
us
Liep
insh
, E
., et
al.
Thi
ored
oxin
fold
as
hom
odim
eriz
atio
n m
odul
e in
the
puta
tive
chap
eron
e E
Rp2
9: N
MR
st
ruct
ures
of t
he d
omai
ns a
nd e
xper
imen
tal
mod
el o
f the
51
kD
a di
mer
. Str
uctu
re 2
001.
v9
p45
7
Silv
enno
inen
, L.
, et a
l. N
MR
str
uctu
re o
f the
N-t
erm
inal
do
mai
n a
of t
he g
lyco
prot
ein
chap
eron
e E
Rp5
7. T
o be
P
ublis
hed.
Kos
tyuc
henk
o, V
.A.,
et a
l. T
he T
ail S
truc
ture
of
Bac
terio
phag
e T
4 an
d its
Mec
hani
sm o
f C
ontr
actio
n.
Nat
.Str
uct.M
ol.B
iol.
2005
. v12
p81
0
Bou
dko,
S.P
., K
uhn,
R.J
., R
ossm
ann,
M.G
. The
Coi
led-
co
il D
omai
n S
truc
ture
of t
he S
in N
ombr
e V
irus
Nuc
leoc
apsi
d P
rote
in.
J.M
ol.B
iol.
2007
. v36
6 p1
538
ER
p57
(N-t
erm
inal
do
mai
n)
synt
hetic
n/
a
phag
e as
sem
bly
phag
e as
sem
bly
Fib
ritin
B
acte
rioph
age
T4
Bac
terio
phag
e 21
BL
Fib
ritin
1V1 H
F
ibrit
in -
fibr
e sh
aft
phag
e co
mpl
ex
asse
mbl
y
1V1 I
F
ibrit
in -
fibr
e sh
aft
phag
e co
mpl
ex
asse
mbl
y
10
x3
F
ibrit
in (
N-t
erm
inal
ph
age
dom
ain)
as
sem
bly
IAA
O
Fib
ritin
del
etio
n m
utan
t ph
age
asse
mbl
y
IAV
Y
Fib
ritin
del
etio
n m
utan
t ph
age
asse
mbl
y
1BF
8 F
imC
ce
ll w
all
orga
niza
tion
and
biog
enes
is
1ZE
3 F
imC
- F
imD
(N
- ce
ll w
all
term
inal
dom
ain)
- or
gani
zatio
n F
imH
com
plex
an
d bi
ogen
esis
IKIU
F
imC
- F
imH
- 0
1-
cell
wal
l m
ethy
l-m
anno
se
orga
niza
tion
com
plex
an
d bi
ogen
esis
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
1.9
Hu
ma
n
Pap
anik
olop
oulo
u, K
., et
al.
Ade
novi
rus
Fib
re S
haft
ad
enov
irus
type
S
eque
nces
Fol
d ln
to th
e N
ativ
e T
riple
Bet
a-S
pira
l F
old
2,
Wh
en
N-T
erm
inal
ly F
used
to t
he B
acte
rioph
age
T4
bact
erio
phag
e F
ibri
tin F
oldo
n T
rimer
isat
ion
Mot
if. J
.Mol
.Bio
l. 20
04. v
342
T4
p2 1
9
1.9
Hu
ma
n
Pap
anik
olop
oulo
u, K
., et
al.
Ade
novi
rus
Fib
re S
haft
ad
enov
irus
type
S
eque
nces
Fol
d ln
to th
e N
ativ
e T
riple
Bet
a-S
pira
l F
old
2,
Whe
n N
-Ter
min
ally
Fus
ed to
the
Bac
teri
opha
ge T
4 ba
cter
ioph
age
Fib
ritin
Fol
don
Trim
eris
atio
n M
otif.
J.M
ol.B
iol.
2004
. v34
2 T4
; A
TC
C V
R-
p2 1
9 8
46
an
d 1
1303
- B
4
2.0
Bac
teri
opha
ge
Bou
dko,
S.P
., et
al.
Des
ign
and
Cry
stal
Str
uctu
re o
f T4
B
acte
riop
hage
T4
Min
i-Fib
ritin
NC
CF
. J.M
ol.B
iol.
2004
v3
39 p
927
2.2
B
acte
riop
hage
E
fimov
, V
.P.,
et a
l. F
ibrit
in e
ncod
ed b
y ba
cter
ioph
age
T4
T4
gene
Wac
has
a p
aral
lel t
riple
- st
rand
ed a
lpha
-hel
ical
co
iled-
coil
stru
ctur
e. J
.Mol
.Bio
l. 19
94. v
242
p470
1.85
B
acte
riop
hage
S
trel
kov,
S.V
., et
al.
Str
uctu
re o
f ba
cter
ioph
age
T4
fibrit
in
T4
M:
a tr
oubl
esom
e pa
ckin
g ar
rang
emen
t. A
cta
Cry
stal
logr
., S
ect.D
199
8. v
54 p
805
nla
E
sche
rich
ia c
oli
Pel
lecc
hia,
M.,
et a
l. N
MR
sol
utio
n st
ruct
ure
of t
he
peri
plas
mic
cha
pero
ne F
imC
. Nat
.Str
uct.B
iol.
1998
. v5
p8
85
1.84
E
sche
richi
a co
li N
ishi
yam
a, M
., et
al.
Str
uctu
ral
basi
s of
cha
pero
ne-
subu
nit
com
plex
rec
ogni
tion
by
the
type
1 p
ilus
asse
mbl
y pl
atfo
rm F
imD
. E
mbo
J. 2
005.
v2
4 p
2075
3.0
E
sche
rich
ia c
oli
Hun
g, C
.S.,
et a
l. S
truc
tura
l bas
is o
f tro
pism
of
Esc
heri
chia
col
i to
the
blad
der
durin
g ur
inar
y tr
act
infe
ctio
n. M
ol.M
icro
biol
. 20
02.
v44
p90
3
1 KLF
IQU
N
11x5
1 QZ
2
1 Q6H
1 Q6U
1 Q61
1 OR
J
Fim
C -
Fim
H -
a-D
- m
anno
se c
ompl
ex
Fim
C -
Fim
H c
ompl
ex
FK
BP
FK
BP
52 (
C-t
erm
inal
do
mai
n) -
Hsp
9O
pept
ide
com
plex
Fkp
A
Fkp
A
Fkp
A -
imm
unos
uppr
essa
nt
FK
506
com
plex
Fla
gella
r ex
port
ch
aper
one
(FIiS
)
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
flage
llum
bi
ogen
esis
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
2.79
2.8
nla
3.0
1.97
2.45
2.25
2.25
Esc
heric
hia
coli
Esc
heric
hia
coli
Met
hano
cocc
us
ther
mol
ithot
roph
ic
us
Hom
o sa
pien
s
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Aqu
ifex
aeol
icus
V
F5
Hun
g, C
.S.,
et a
l. S
truc
tura
l bas
is o
f tro
pism
of
Esc
heric
hia
coli
to th
e bl
adde
r du
ring
urin
ary
trac
t in
fect
ion.
Mol
.Mic
robi
ol. 2
002.
v44
p90
3
Cho
udhu
ry,
D.,
et a
l. X
-ray
str
uctu
re o
f the
Firn
C-F
imH
ch
aper
one-
adhe
sin
com
plex
from
uro
path
ogen
ic
Esc
heric
hia
coli.
Sci
ence
199
9. v
285
p106
1
Suz
uki,
R.,
et a
l. T
hree
-dim
ensi
onal
Sol
utio
n S
truc
ture
of
an
Arc
haea
l F
KB
P w
ith a
Dua
l Fun
ctio
n of
Pep
tidyl
Pro
lyl
cis-
tran
s ls
omer
ase
and
Cha
pero
ne-li
ke A
ctiv
ities
. J.
Mol
.Bio
l. 20
03.
v328
p11
49
Wu,
B.,
et a
l. 3D
str
uctu
re o
f hu
man
FK
506-
bind
ing
prot
ein
52:
Impl
icat
ions
for
the
asse
mbl
y of
the
gluc
ocor
ticoi
d re
cept
orlH
sp90
limm
unop
hilin
he
tero
com
plex
. P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
4. v
l01
p8
348
Sau
l, F
.A.,
et a
l. S
truc
tura
l an
d F
unct
iona
l Stu
dies
of
Fkp
A fr
om E
sche
richi
a co
li, a
cis
ltran
s P
eptid
yl-p
roly
l ls
omer
ase
with
Cha
pero
ne A
ctiv
ity.
J.M
ol.B
iol.
2004
. v3
35 p
595
Sau
l, F
.A.,
et a
l. S
truc
tura
l and
Fun
ctio
nal S
tudi
es o
f F
kpA
from
Esc
heric
hia
coli,
a c
isltr
ans
Pep
tidyl
-pro
lyl
lsom
eras
e w
ith C
hape
rone
Act
ivity
. J.
Mol
.Bio
l. 20
04.
v335
p59
5
Sau
l, F
.A.,
et a
l. S
truc
tura
l and
Fun
ctio
nal S
tudi
es o
f F
kpA
from
Esc
heric
hia
coli,
a c
isltr
ans
Pep
tidyl
-pro
lyl
lsom
eras
e w
ith C
hape
rone
Act
ivity
. J.
Mol
.Bio
l. 20
04.
v335
p59
5
Evd
okim
ov,
A.G
., et
al.
Sim
ilar
mod
es o
f po
lype
ptid
e re
cogn
ition
by
expo
rt c
hape
rone
s in
flag
ella
r bi
osyn
thes
is a
nd ty
pe I
ll se
cret
ion.
Nat
.Str
uct.B
iol.
2003
. v1
0 p7
89
1 OR
Y
2GA
5
1 NB
W
1 G3
l
1 GR
5
1 GR
L
1 J4Z
1 KP
O
1 SS
8
1 XC
K
Fla
gella
r ex
port
ch
aper
one
(FliS
) -
flage
llin
(FIiC
) co
mpl
ex
flage
llum
bi
ogen
esis
X
-ray
NM
R
X-r
ay
X-r
ay
Cry
o-
EM
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Aqu
ifex
aeol
icus
V
F5
Evd
okim
ov,
A.G
., et
al.
Sim
ilar
mod
es o
f po
lype
ptid
e re
cogn
ition
by
expo
rt c
hape
rone
s in
flag
ella
r bi
osyn
thes
is a
nd ty
pe I
ll se
cret
ion.
Nat
.Str
uct.B
iol.
2003
. v1
0 p7
89
Fra
taxi
n m
etal
ion
tran
spor
t S
acch
arom
yces
ce
revi
siae
H
e, Y
., et
al.
Yea
st F
rata
xin
Sol
utio
n S
truc
ture
, Ir
on
Bin
ding
and
Fer
roch
elat
ase
Inte
ract
ion.
Bio
chem
istr
y 20
04.
v43
~1
62
54
Gly
cero
l deh
ydra
tase
re
activ
ase
prot
ein
reac
tivat
ion
Kle
bsie
lla
pneu
mon
iae
Liao
, D
.4,
et a
l. S
truc
ture
of g
lyce
rol d
ehyd
rata
se
reac
tivas
e: A
new
type
of
mol
ecul
ar c
hape
rone
. S
truc
ture
200
3. v
l 1 p
109
prot
ein
fold
ing,
co
-cha
pero
ne
to G
roE
L
Bac
terio
phag
e T4
H
unt,
J.F
., et
al.
Str
uctu
ral a
dapt
atio
ns in
the
spec
ializ
ed
bact
erio
phag
e T
4 co
-cha
pero
nin
Gp3
1 ex
pand
the
size
of
the
Anf
inse
n ca
ge. C
ell
1997
. v90
p36
1
Gro
EL
prot
ein
fold
ing
Esc
heri
chia
co
li R
anso
n, N
.A.,
et a
l. A
TP
-bou
nd s
tate
s of
Gro
EL
capt
ured
by
cryo
-ele
ctro
n m
icro
scop
y. C
ell 2
001.
v10
7 p8
69
Gro
EL
prot
ein
fold
ing
Esc
heric
hia
coli
Bra
ig,
K.,
et a
l. T
he c
ryst
al s
truc
ture
of t
he b
acte
rial
chap
eron
in G
roE
L at
2.8
A.
Nat
ure
1994
. v37
1 p5
78
Esc
heric
hia
coli
Wan
g, J
., B
oisv
ert,
D.C
. A
Gro
ELI
Gro
ES
com
plex
G
roE
L pr
otei
n fo
ldin
g st
ruct
ure
revi
site
d: th
e st
ruct
ure-
base
d m
echa
nism
of
AT
P h
ydro
lysi
s. T
o be
pub
lishe
d.
Wan
g, J
., B
oisv
ert,
D.C
. A G
roE
LIG
roE
S c
ompl
ex
stru
ctur
e re
visi
ted:
the
str
uctu
re-b
ased
mec
hani
sm o
f A
TP
hyd
roly
sis.
To
be P
ublis
hed.
Gro
EL
Esc
heric
hia
coli
prot
ein
fold
ing
Gro
EL
Esc
heric
hia
coli
prot
ein
fold
ing
prot
ein
fold
ing
Cha
udhr
y, C
., et
al.
Exp
lorin
g th
e st
ruct
ural
dyn
amic
s of
th
e E
.col
i cha
pero
nin
Gro
EL
usin
g tr
ansl
atio
n-lib
ratio
n-
scre
w c
ryst
allo
grap
hic
refin
emen
t of
inte
rmed
iate
sta
tes.
J.
Mol
.Bio
l. 20
04.
v342
p22
9
Gro
EL
Esc
heric
hia
coli
Bar
tolu
cci,
C.,
et a
l. C
ryst
al s
truc
ture
of w
ild-t
ype
chap
eron
in G
roE
L. J
.Mol
.Bio
l. 20
05.
v354
p94
0
2NW
C
Gro
EL
prot
ein
fold
ing
X-r
ay
X-r
ay
Cry
o-
EM
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Cry
o-
EM
Cry
o-
EM
Esc
heric
hia
coli
Kis
er,
P.D
., Lo
dow
ski,
D.T
., P
alcz
ewsk
i, K
. P
urifi
catio
n,
crys
talli
zatio
n an
d st
ruct
ure
dete
rmin
atio
n of
nat
ive
Gro
EL
from
Esc
heric
hia
coli
lack
ing
boun
d po
tass
ium
io
ns. A
cta
Cry
stal
logr
.,Sec
t.F 2
007.
v63
p45
7
1 KP
8 G
roE
L -
AT
P
prot
ein
fold
ing
Esc
heric
hia
coli
Wan
g, J
., B
oisv
ert,
D.C
. Str
uctu
ral B
asis
for
Gro
EL-
as
sist
ed P
rote
in F
oldi
ng fr
om th
e C
ryst
al S
truc
ture
of
(Gro
EL-
KM
gAT
P)1
4 at
2.0
A R
esol
utio
n. J
.Mol
.Bio
l. 20
03.
v327
p84
3
Cla
re,
D.K
., et
al.
An
Exp
ande
d P
rote
in F
oldi
ng C
age
in
the
Gro
el-G
p31
Com
plex
. J.M
ol.B
iol.
2006
. v35
8 p9
05
prot
ein
fold
ing
prot
ein
fold
ing
Bac
terio
phag
e T4
2C
GT
1 PC
Q
1 PF
9
1 SX
4
1 AO
N
2C7D
2C7C
Gro
EL
- AT
P -
Gp3
1 co
mpl
ex
Gro
EL
- Gro
ES
- A
DP
co
mpl
ex
Esc
heric
hia
coli
Cha
udhr
y, C
., et
al.
Rol
e of
the
gam
ma-
phos
phat
e of
A
TP
in tr
igge
ring
prot
ein
fold
ing
by
Gro
EL-
Gro
ES
: fu
nctio
n, s
truc
ture
and
ene
rget
ics.
EM
BO
J.
2003
. v22
p4
877
Gro
EL
- Gro
ES
- A
DP
co
mpl
ex
Cha
udhr
y, C
., et
al.
Rol
e of
the
gam
ma-
phos
phat
e of
A
TP
in
trig
gerin
g pr
otei
n fo
ldin
g b
y G
roE
L-G
roE
S:
func
tion,
str
uctu
re a
nd e
nerg
etic
s. E
MB
O J
. 20
03. v
22
p487
7
prot
ein
fold
ing
Esc
heric
hia
coli
Gro
EL
- Gro
ES
- A
DP
, pr
otei
n fo
ldin
g E
sche
richi
a co
li C
haud
hry,
C.,
et a
l. E
xplo
ring
the
stru
ctur
al d
ynam
ics
of
the
E.c
oli c
hape
roni
n G
roE
L us
ing
tran
slat
ion-
libra
tion-
sc
rew
cry
stal
logr
aphi
c re
finem
ent o
f in
term
edia
te s
tate
s.
J.M
ol.B
iol.
2004
. v34
2 p2
29
Gro
EL
- G
roE
S -
AD
P,
com
plex
pr
otei
n fo
ldin
g
prot
ein
fold
ing
prot
ein
fold
ing
Esc
heric
hia
coli
Xu,
Z.,
Hor
wic
h, A
.L.,
Sig
ler,
P.B
. The
cry
stal
str
uctu
re o
f th
e as
ymm
etric
Gro
EL-
Gro
ES
-(A
DP
)7 c
hape
roni
n co
mpl
ex.
Nat
ure
1997
. v38
8 p7
41
Gro
EL
- Gro
ES
- A
DP
, co
mpl
ex
Esc
heric
hia
coli
Esc
heric
hia
coli
Ran
son,
N.A
., et
al.
Allo
ster
ic S
igna
lling
of
AT
P
Hyd
roly
sis
in G
roel
-Gro
es C
ompl
exes
. N
at.S
truc
t.Mol
.Bio
l. 20
06. v
13 p
147
Gro
EL
- Gro
ES
- AT
P,
com
plex
R
anso
n, N
.A.,
et a
l. A
llost
eric
Sig
nalli
ng o
f A
TP
H
ydro
lysi
s in
Gro
el-G
roes
Com
plex
es.
Nat
.Str
uct.M
ol.B
iol.
2006
. v1
3 p1
47
1 EG
S
1 MN
F
1 DK
7
1 JO
N
1 LA
1
1 SR
V
1 DK
D
1 FY
9
1 FY
A
1 KID
Gro
EL
- Gro
ES
pe
ptid
e co
mpl
ex
Gro
EL
- pe
ptid
e co
mpl
ex
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
n)
- dod
ecam
eric
pep
tide
com
plex
Gro
EL
(api
cal d
omai
n,
mut
ant)
Gro
EL
(api
cal d
omai
n,
mut
ant)
Gro
EL
(api
cal d
omai
n,
mut
ant)
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
n/a
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
The
rmus
th
erm
ophi
lus
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Land
ry, S
.J.,
et a
l. In
terp
lay
of s
truc
ture
and
dis
orde
r in
co
chap
eron
in m
obile
loop
s. P
roc.
Nat
l.Aca
d.S
ci.U
SA
19
96. v
93 p
1162
2
Wan
g, J
., C
hen,
L.
Dom
ain
Mot
ions
in G
roE
L up
on
Bin
ding
of
an
Olig
opep
tide.
J.M
ol.B
iol.
2003
. v3
34 p
489
Che
n, L
., S
igle
r, P
.B. T
he c
ryst
al s
truc
ture
of
a G
roE
LJpe
ptid
e co
mpl
ex: p
last
icity
as
a ba
sis
for
subs
trat
e di
vers
ity.
Cel
l 199
9. v
99 p
75
7
Zah
n, R
., et
al.
Cha
pero
ne a
ctiv
ity a
nd s
truc
ture
of
mon
omer
ic p
olyp
eptid
e bi
ndin
g do
mai
ns o
f G
roE
L.
Pro
c.N
atl.A
cad.
Sci
.US
A 1
996.
v93
p15
024
Ash
crof
t, A
.E.,
et a
l. S
truc
tura
l pla
stic
ity a
nd n
onco
vale
nt
subs
trat
e bi
ndin
g in
the
Gro
EL
apic
al d
omai
n. A
stu
dy
usin
g el
ectr
ospa
y io
niza
tion
mas
s sp
ectr
omet
ry a
nd
fluor
esce
nce
bind
ing
stud
ies.
J.B
iol.C
hem
. 20
02. v
277
p331
15
Wal
sh,
M.A
., et
al.
Tak
ing
MA
D to
the
ext
rem
e: u
ltraf
ast
prot
ein
stru
ctur
e de
term
inat
ion.
Act
a C
ryst
allo
gr.,
Sec
t.D
1999
. v55
p1
I68
Che
n, L
., S
igle
r, P
.B. T
he c
ryst
al s
truc
ture
of
a G
roE
LJpe
ptid
e co
mpl
ex: p
last
icity
as
a ba
sis
for
subs
trat
e di
vers
ity.
Cel
l 199
9. v
99 p
75
7
Wan
g, Q
., B
uckl
e, A
.M.,
Fer
sht,
A.R
. S
tabi
lizat
ion
of
Gro
EL
min
icha
pero
nes
by c
ore
and
surf
ace
mut
atio
ns.
J.M
ol.B
iol.
2000
. v2
98 p
917
Wan
g, Q
., B
uckl
e, A
.M.,
Fer
sht,
A.R
. S
tabi
lizat
ion
of
Gro
EL
min
icha
pero
nes
by c
ore
and
surf
ace
mut
atio
ns.
J.M
ol.B
iol.
2000
. v29
8 p9
17
Buc
kle,
A.M
., Z
ahn,
R.,
Fer
sht,
A.R
. A
str
uctu
ral m
odel
fo
r G
roE
L-po
lype
ptid
e re
cogn
ition
. P
roc.
Nat
l.Aca
d.S
ci. U
SA
199
7. v
94 p
3571
IOE
L
Gro
EL
(mut
ant)
pr
otei
n fo
ldin
g X
-ray
E
sche
richi
a co
li B
raig
, K.,
Ada
ms,
P.D
., B
rung
er, A
.T.
Con
form
atio
nal
varia
bilit
y in
the
refin
ed s
truc
ture
of t
he c
hape
roni
n G
roE
L at
2.8
A r
esol
utio
n. N
at.S
truc
t.Bio
l. 19
95. v
2 p1
083
2EU
1 G
roE
L (m
utan
t)
Cab
o-B
ilbao
, A,,
et a
l. C
ryst
al s
truc
ture
of t
he
tem
pera
ture
-sen
sitiv
e an
d al
lost
eric
-def
ectiv
e ch
aper
onin
Gro
EL(
E46
1 K).
J.S
truc
t.Bio
l. 20
06.
v155
p4
82
prot
ein
fold
ing
X-r
ay
Esc
heric
hia
coli
2C7E
G
roE
L (m
utan
t) -
ATP
, pr
otei
n fo
ldin
g co
mpl
ex
Cry
o-
EM
E
sche
richi
a co
li
Esc
heric
hia
coli
Esc
heric
hia
coli
Ran
son,
N.A
., et
al.
AT
P-B
ound
Sta
tes
of G
roel
C
aptu
red
by C
ryo-
Ele
ctro
n M
icro
scop
y. C
ell 2
001.
v10
7 p8
69
IGR
U
Gro
EL-
(AT
P),
-
prot
ein
fold
ing
Gro
ES
-(A
DP
),
com
plex
Cry
o-
EM
R
anso
n, N
.A.,
et
al. A
TP
-bou
nd s
tate
s of
Gro
EL
capt
ured
by
cryo
-ele
ctro
n m
icro
scop
y. C
ell 2
00 1
. v10
7 p8
69
1SX
3 G
roE
L,,
- (A
TP
yS),,
pr
otei
n fo
ldin
g C
haud
hry,
C.,
et a
l. E
xplo
ring
the
stru
ctur
al d
ynam
ics
of
the
E.c
oli c
hape
roni
n G
roE
L us
ing
tran
slat
ion-
libra
tion-
sc
rew
cry
stal
logr
aphi
c re
finem
ent
of in
term
edia
te s
tate
s.
J.M
ol.B
iol.
2004
. v3
42 p
229
Cha
udhr
y, C
., e
t al
. Exp
lorin
g th
e st
ruct
ural
dyn
amic
s of
th
e E
.col
i cha
pero
nin
Gro
EL
usin
g tr
ansl
atio
n-lib
ratio
n-
scre
w c
ryst
allo
grap
hic
refin
emen
t of
inte
rmed
iate
sta
tes.
J.
Mol
.Bio
l. 20
04.
v342
p22
9
X-r
ay
ISV
T
Gro
EL,
, -
Gro
ES
, -
prot
ein
fold
ing
(AD
P-A
I Fx)
, E
sche
richi
a co
li X
-ray
2FY
P
Grp
94 -
rad
este
r pr
otei
n fo
ldin
g,
amin
e co
mpl
ex
ER
par
alog
of
Hsp
9O
X-r
ay
Can
is fa
mili
aris
Im
mor
min
o, R
.M.,
Gew
irth,
D.T
., B
lagg
, B.S
. In
hibi
ttory
Li
gand
s A
dopt
Diff
eren
t C
onfo
rmat
ions
Whe
n B
ound
to
Hsp
9O o
r G
RP
94:
Impl
icat
ions
for
Par
alog
-spe
cific
Dru
g D
esig
n. T
o be
pub
lishe
d.
IYT
O
Grp
94 (
mut
ant)
pr
otei
n fo
ldin
g,
ER
par
alog
of
Hsp
9O
X-r
ay
Can
is fa
mili
aris
D
ollin
s, D
.E.,
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T. S
truc
ture
of
Unl
igan
ded
GR
P94
, th
e E
ndop
lasm
ic R
etic
ulum
H
sp9O
: bas
is fo
r nu
cleo
tide-
indu
ced
conf
orm
atio
nal
chan
ge.
J.B
iol.C
hem
. 20
05.
v280
p30
438
1 YT
1
1 YT
2
IYS
Z
1 QY
E
2H8M
2ES
A
2EX
L
2GQ
P
2HC
H
Grp
94 (
mut
ant)
- A
DP
co
mpl
ex
Grp
94 (
mut
ant)
- A
DP
co
mpl
ex
Grp
94 (
mut
ant)
- N
EC
A c
ompl
ex
Grp
94 (
N-t
erm
inal
do
mai
n) -
2-
chlo
rodi
deox
yade
nosi
n e
com
plex
Grp
94 (
N-t
erm
inal
do
mai
n) -
2-lo
do-
NE
CA
com
plex
Grp
94 (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
Grp
94 (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
com
plex
Grp
94 (
N-t
erm
inal
do
mai
n) -
ligan
d co
mpl
ex
Grp
94 (
N-t
erm
inal
do
mai
n) -
liga
nd
com
plex
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp91
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Can
is fa
rnili
aris
Can
is fa
mili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Dol
lins,
D.E
., Im
mor
min
o, R
.M.,
Gew
irth,
D.T
. Str
uctu
re
of U
nlig
ande
d G
RP
94, t
he E
ndop
lasm
ic R
etic
ulum
H
sp9O
: bas
is fo
r nu
cleo
tide-
indu
ced
conf
orm
atio
nal
chan
ge. J
.Bio
l.Che
m. 2
005.
v28
0 p3
0438
Dol
lins,
D.E
., Im
mor
min
o, R
.M.,
Gew
irth,
D.T
. Str
uctu
re
of U
nlig
ande
d G
RP
94, t
he E
ndop
lasm
ic R
etic
ulum
H
sp9O
: bas
is fo
r nu
cleo
tide-
indu
ced
conf
orm
atio
nal
chan
ge. J
.Bio
l.Che
m.
2005
. v2
80 p
3043
8
Dol
lins,
D.E
., Im
mor
min
o, R
.M.,
Gew
irth,
D.T
. Str
uctu
re
of U
nlig
ande
d G
RP
94, t
he E
ndop
lasm
ic R
etic
ulum
H
sp9O
: bas
is fo
r nu
cleo
tide-
indu
ced
conf
orm
atio
nal
chan
ge. J
.Bio
l.Che
m. 2
005.
v28
0 p3
0438
Sol
dano
, K
.L.,
et a
l. S
truc
ture
of t
he N
-ter
min
al D
omai
n of
GR
P94
. B
asis
for
Liga
nd S
peci
ficity
and
Reg
ulat
ion.
J.
Bio
l.Che
m.
2003
. v27
8 p4
8330
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T. N
-Dom
ain
Of G
rp94
In
Com
plex
With
the
2-lo
do-N
EC
A. T
o be
pub
lishe
d.
Imm
orm
ino,
R.M
., et
al.
Cry
stal
Str
uctu
re o
f G
RP
94 w
ith
the
spec
ific
mut
atio
n K
S16
8-16
9AA
; with
bou
nd
Gel
dana
myc
in. T
o be
pub
lishe
d.
Imm
orm
ino,
R.M
., et
al.
GR
P94
N-t
erm
inal
Dom
ain
boun
d to
gel
dana
myc
in. T
o be
pub
lishe
d.
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T. A
dens
ine
Sca
ffold
in
hibi
tors
of
GR
P94
. To
be p
ublis
hed.
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T. N
-Dom
ain
Of G
rp94
In
Com
plex
With
the
Nov
el L
igan
d N
-am
inoe
thyl
C
arbo
xyam
ido
Ade
nosi
ne.
To
be p
ublis
hed.
2HG
1
1 QY
5
1 UO
Y
2GF
D
1 QY
8
1 U2
0
1 uoz
1 TC
6
ITB
W
Grp
94
(N
-ter
min
al
do
ma
in)
- lig
and
com
plex
Grp
94
(N
-ter
min
al
do
ma
in) - N
EC
A
com
plex
Grp
94
(N
-ter
min
al
do
ma
in)
- N
-Pro
pyl
Car
boxy
amid
o A
deno
sine
com
plex
do
ma
in)
- ra
dam
ide
com
plex
Grp
94
(N
-ter
min
al
do
ma
in)
- rad
icic
ol
com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mis
sing
ch
arge
d do
mai
n) -
NE
CA
com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mis
sing
ch
arge
d do
mai
n) -
radi
cico
l com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mut
ant)
- A
DP
Grp
94
(N
-ter
min
al
dom
ain,
mut
ant)
- A
MP
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp92
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
2.3
Can
is fa
mili
aris
1.75
C
anis
fam
iliar
is
2.3
Can
is fa
mili
aris
2.3
Can
is fa
mili
aris
1.85
C
anis
fam
iliar
is
2.1
C
anis
fam
iliar
is
1.9
Can
is fa
mili
aris
1.87
C
anis
fam
iliar
is
2.15
C
anis
fam
iliar
is
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T.
N-D
omai
n O
f Grp
94 I
n
Com
plex
With
the
Nov
el L
igan
d N
-(2-
hydr
oxy1
)eth
yl
Car
boxy
amid
o A
deno
sine
. T
o be
pub
lishe
d.
Sol
dano
, K
.L.,
et a
l. S
truc
ture
of t
he N
-ter
min
al d
omai
n of
GR
P94
. B
as
s fo
r lig
and
spec
ifici
ty a
nd r
egul
atio
n.
J.B
iol.C
hem
. 20
03.
v278
p48
330
Gew
irth,
D.T
., Im
mor
min
o, R
.M.
N-D
omai
n O
f Grp
94,
with
the
Cha
rged
Dom
ain,
In
Com
plex
W~
th the
Nov
el
Liga
nd N
-Pro
pyl C
arbo
xyam
ido
Ade
nosi
ne.
To
be
publ
ishe
d.
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T.,
Bla
gg, B
.S.
Inhi
bitto
ry
Liga
nds
Ado
pt D
iffer
ent C
onfo
rmat
ions
Whe
n B
ound
to
Hsp
9O o
r G
RP
94:
Impl
icat
ions
for
Par
alog
-spe
cific
Dru
g D
esig
n. T
o be
pub
lishe
d.
Sol
dano
, K
.L.,
et a
l. S
truc
ture
of t
he N
-ter
min
al d
omai
n of
GR
P94
. B
asis
for
ligan
d sp
ecifi
city
and
reg
ulat
ion.
J.
Bio
l.Che
m.
2003
. v2
78 p
4833
0
Sol
dano
, K
.L.,
et a
l. S
truc
ture
of t
he N
-ter
min
al d
omai
n of
GR
P94
. B
asis
for
ligan
d sp
ecifi
city
and
reg
ulat
ion.
J.
Bio
l.Che
m.
2003
. v2
78 p
4833
0
Gew
irth,
D.T
., Im
mor
min
o, R
.M. N
-Dom
ain
Of G
rp94
La
ckin
g T
he C
harg
ed D
omai
n In
Com
plex
With
R
adic
icol
. To
be p
ublis
hed.
Imm
orm
ino,
R.M
., et
al.
Liga
nd-in
duce
d C
onfo
rmat
iona
l S
hift
in th
e N
-ter
min
al D
omai
n of
GR
P94
, an
Hsp
9O
Cha
pero
ne.
J.B
iol.C
hem
. 20
04.
v279
p46
162
Imm
orm
ino,
R.M
., et
al.
Liga
nd-in
duce
d C
onfo
rmat
iona
l S
hift
in th
e N
-ter
min
al D
omai
n of
GR
P94
, an
Hsp
9O
Cha
pero
ne.
J.B
iol.C
hem
. 20
04.
v279
p46
162
1 TC
O
1 TL5
1 FEO
I FE
E
1 TL4
1 FE
4
2JO
P
2JO
R
1 HK
9
Grp
94 (
N-t
erm
inal
pr
otei
n fo
ldin
g,
dom
ain,
mut
ant)
- A
TP
E
R p
aral
og o
f H
sp9O
Ha
hl
met
al io
n tr
ansp
ort
Ha
hl
- cd
2'
met
al io
n tr
ansp
ort
Ha
hl
- C
u'
met
al io
n tr
ansp
ort
Ha
hl -
Cu'
m
etal
ion
tran
spor
t
Ha
hl
- H
~"
m
etal
ion
tran
spor
t
hem
e tr
ansp
ort
hem
e tr
ansp
ort
regu
latio
n of
tr
ansc
riptio
n,
RN
A
chap
eron
e
X-r
ay
NM
R
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
2.2
Can
isfa
mili
aris
nla
H
omo
sapi
ens
1.75
H
omo
sapi
ens
1.8
Ho
mo
sap
iens
nla
H
omo
sapi
ens
1.75
H
omo
sapi
ens
1.7
Yer
sini
a en
tero
colit
ica
1.9
Yer
sini
a en
tero
colit
ica
2.15
E
sche
richi
a co
li
Imm
orm
ino,
R.M
., et
al.
Liga
nd-in
duce
d C
onfo
rrna
tiona
l S
hift
in t
he N
-ter
min
al D
omai
n of
GR
P94
, an
Hsp
9O
Cha
pero
ne.
J.B
iol.C
hem
. 20
04. v
279
p461
62
Ana
stas
sopo
ulou
, I.,
et
al.
Sol
utio
n S
truc
ture
of
the
Apo
an
d C
oppe
r(1)
-Loa
ded
Hum
an M
etal
loch
aper
one
HA
H 1
. B
ioch
emis
try
2004
. v4
3 ~
13
04
6
Wer
nirn
ont,
A.K
., et
al.
Str
uctu
ral b
asis
for
copp
er
tran
sfer
by
the
met
allo
chap
eron
e fo
r th
e M
enke
sNV
ilson
di
seas
e pr
otei
ns.
Nat
.Str
uct.B
iol.
2000
. v7
p76
6
Wer
nirn
ont,
A.K
., et
al.
Str
uctu
ral b
asis
for
copp
er
tran
sfer
by
the
met
allo
chap
eron
e fo
r th
e M
enke
sNV
ilson
di
seas
e pr
otei
ns.
Nat
.Str
uct.B
iol.
2000
. v7
p76
6
Ana
stas
sopo
ulou
, I.,
et
al. S
olut
ion
Str
uctu
re o
f th
e A
po
and
Cop
per(
1)-L
oade
d H
uman
Met
allo
chap
eron
e H
AH
1.
Bio
chem
istr
y 20
04.
v43
p130
46
Wer
nirn
ont,
A.K
., et
al.
Str
uctu
ral b
asis
for
copp
er
tran
sfer
by
the
rnet
allo
chap
eron
e fo
r th
e M
enke
sNV
ilson
di
seas
e pr
otei
ns.
Nat
.Str
uct.B
iol.
2000
. v7
p76
6
Sch
neid
er, S
., et
al.
An
lndu
ced
Fit
Con
forr
natio
nal
Cha
nge
Und
erlie
s th
e B
indi
ng M
echa
nism
of t
he H
eme
Tra
nspo
rt P
rote
obac
teria
-Pro
tein
Hem
s. J
.Bio
l.Che
m.
2006
. v2
81 p
3260
6
Sch
neid
er, S
., et
al.
An
lndu
ced
Fit
Con
forr
natio
nal
Cha
nge
Und
erlie
s th
e B
indi
ng M
echa
nism
of t
he H
eme
Tra
nspo
rt P
rote
obac
teria
-Pro
tein
Hem
s. J
.Bio
l.Che
m.
2006
. v2
81 p
3260
6
Sau
ter,
C.,
Bas
quin
, J.,
Suc
k, D
. Sm
-Lik
e P
rote
ins
in
Eub
acte
ria:
The
Cry
stal
Str
uctu
re o
f th
e H
fq P
rote
in fr
om
Esc
heric
hia
Col
i. N
ucle
ic A
cids
Res
. 200
3. v
31 p
4091
1 ELW
1 ELR
1 S4Z
1 FP
O
1 AT
R
HO
P (
TP
RI
dom
ain)
- H
sc70
pep
tide
com
plex
HO
P (
TP
R2A
dom
ain)
-
Hsp
9O p
eptid
e co
mpl
ex
HP
I (s
hado
w d
omai
n)
- C
AF
-1 (
PX
VX
L m
otif)
Hsc
2O (
Hsc
B)
Hsc
70 -
AD
P
1 AT
S
Hsc
70 -
AD
P
Hsc
70 (
AT
Pas
e do
mai
n)
prot
ein
fold
ing,
ad
apto
r pr
otei
n to
hs
p70
and
hsp9
0
prot
ein
fold
ing,
ad
apto
r pr
otei
n to
hs
p70
and
hsp9
0
chro
mat
in
asse
mbl
y
prot
ein
fold
ing,
a
J-ty
pe c
o-
chap
eron
e to
E
.col
i Hsc
A
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0
X-r
ay
1.6
Hom
o sa
pien
s S
cheu
fler,
C.,
et a
l. S
truc
ture
of T
PR
dom
ain-
pept
ide
com
plex
es:
criti
cal e
lem
ents
in th
e as
sem
bly
of t
he
Hsp
70-H
sp9O
mul
ticha
pero
ne m
achi
ne.
Cel
l 200
0. v
lOl
p199
X-r
ay
1.9
Hom
o sa
pien
s S
cheu
fler,
C.,
et a
l. S
truc
ture
of T
PR
dom
ain-
pept
ide
com
plex
es:
criti
cal e
lem
ents
in th
e as
sem
bly
of t
he
Hsp
70-H
sp9O
mul
ticha
pero
ne m
achi
ne.
Cel
l 200
0. v
lOl
p199
NM
R
nla
M
us m
uscu
lus
Thi
ru, A
., et
al.
Str
uctu
ral b
asis
of H
PII
PX
VX
L m
otif
pept
ide
inte
ract
ions
and
HP
I lo
calis
atio
n to
he
tero
chro
mat
in.
EM
BO
J. 2
004.
v23
p48
9
X-r
ay
1.8
Esc
heric
hia
coli
Cup
p-V
icke
ry,
J.R
., V
icke
ry,
L.E
. C
ryst
al s
truc
ture
of
Hsc
20, a
J-t
ype
Co-
chap
eron
e fr
om E
sche
richi
a co
li.
J.M
ol.B
iol.
2000
. v30
4 p8
35
X-r
ay
2.34
B
os ta
urus
O
'Brie
n, M
.C.,
McK
ay,
D.B
. Thr
eoni
ne 2
04 o
f the
ch
aper
one
prot
ein
Hsc
70 in
fluen
ces
the
stru
ctur
e of
the
activ
e si
te,
but
is n
ot e
ssen
tial f
or A
TP
hyd
roly
sis.
J.
Bio
l.Che
m.
1993
. v26
8 p2
4323
X-r
ay
2.43
B
os ta
urus
O
'Brie
n, M
.C.,
McK
ay,
D.B
. Thr
eoni
ne 2
04 o
f the
ch
aper
one
prot
ein
Hsc
70 in
fluen
ces
the
stru
ctur
e of
the
ac
tive
site
, bu
t is
not
ess
entia
l for
AT
P h
ydro
lysi
s.
J.B
iol.C
hem
. 19
93. v
268
p243
23
X-r
ay
1.93
B
os ta
urus
F
lahe
rty,
K.M
., D
eLuc
a-F
lahe
rty,
C.,
McK
ay,
D.B
. T
hree
- di
men
sion
al s
truc
ture
of t
he A
TP
ase
frag
men
t of
a 7
0K
heat
-sho
ck c
ogna
te p
rote
in. N
atur
e 19
90. v
346
p623
hom
olog
1 HP
M
Hsc
70 (A
TP
ase
prot
ein
fold
ing
X-r
ay
dom
ain)
- A
DP
1 HX
1 H
sc70
(AT
Pas
e pr
otei
n fo
ldin
g,
X-r
ay
dom
ain)
- B
AG
dom
ain
Gro
ES
-like
co
mpl
ex
1 QQ
M
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
IQQ
N
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
IQQ
O
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
2BU
P
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
1 KA
X
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
- A
DP
prot
ein
fold
ing,
X
-ray
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
X
-ray
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
X
-ray
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
X
-ray
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
X
-ray
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
1.7
60
s ta
urus
1.9
Hom
o sa
pien
s
1.9
60
s ta
urus
1.9
60
s ta
urus
1.9
60
s ta
urus
1 .7
60
s ta
urus
1.7
60
s ta
urus
Wilb
anks
, S
.M.,
McK
ay, D
.B. H
ow p
otas
sium
affe
cts
the
activ
ity o
f the
mol
ecul
ar c
hape
rone
Hsc
70. I
I. P
otas
sium
bi
nds
spec
ifica
lly in
the
AT
Pas
e ac
tive
site
. J.B
iol.C
hem
. 19
95. v
270
p225
1
Son
derm
ann,
H.,
et a
l. S
truc
ture
of a
Bag
IHsc
70
com
plex
: con
verg
ent f
unct
iona
l evo
lutio
n of
Hsp
70
nucl
eotid
e ex
chan
ge fa
ctor
s. S
cien
ce 2
001.
v29
1 p1
553
John
son,
E.R
., M
cKay
, D.B
. M
appi
ng th
e ro
le o
f ac
tive
site
res
idue
s fo
r tr
ansd
ucin
g an
AT
P-in
duce
d co
nfor
mat
iona
l cha
nge
in th
e bo
vine
70-
kDa
heat
sho
ck
cogn
ate
prot
ein.
Bio
chem
istr
y 19
99. v
38 p
1082
3
John
son,
E.R
., M
cKay
, D.B
. Map
ping
the
rol
e of
act
ive
site
res
idue
s fo
r tr
ansd
ucin
g an
AT
P-in
duce
d co
nfor
mat
iona
l cha
nge
in th
e bo
vine
70-
kDa
heat
sho
ck
cogn
ate
prot
ein.
Bio
chem
istr
y 19
99. v
38 ~
10
82
3
John
son,
E.R
., M
cKay
, D.B
. Map
ping
the
role
of
activ
e si
te r
esid
ues
for
tran
sduc
ing
an A
TP
-indu
ced
conf
orm
atio
nal c
hang
e in
the
bovi
ne 7
0-kD
a he
at s
hock
co
gnat
e pr
otei
n. B
ioch
emis
try
1999
. v38
p10
823
Sou
sa, M
.C.,
McK
ay, D
.B. T
he h
ydro
xyl o
f thr
eoni
ne 1
3 of
the
bovi
ne 7
0-kD
a he
at s
hock
cog
nate
pro
tein
is
esse
ntia
l for
tra
nsdu
cing
the
AT
P-in
duce
d co
nfor
mat
iona
l cha
nge.
Bio
chem
istr
y 19
98. v
37 p
1539
2
O'B
rien,
M.C
., F
lahe
rty,
K.M
., M
cKay
, D.B
. Lys
ine
71 o
f th
e ch
aper
one
prot
ein
Hsc
70 Is
ess
entia
l for
AT
P
hydr
olys
is. J
.Bio
l.Che
m.
1996
. v27
1 p1
5874
1 KA
Y
IKA
Z
1 UD
O
IYU
W
1 CK
R
7HS
C
Hsc
70 (
AT
Pas
e do
mai
n, m
utan
t) -
AD
P
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
- A
DP
Hsc
70 (
C-t
erm
inal
10
kDa
subd
omai
n)
Hsc
70 (m
utan
t)
Hsc
70 (
subs
trat
e bi
ndin
g do
mai
n)
Hsc
70 (
subs
trat
e bi
ndin
g do
mai
n)
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a
cons
titut
ivel
y ex
pres
sed
hsp7
0 ho
mol
og
prot
ein
fold
ing,
a c
onst
itutiv
ely
expr
esse
d hs
p70
hom
oloa
X-r
ay
1.7
60
s ta
urus
X-r
ay
1.7
Bos
taur
us
X-r
ay
3.45
R
attu
s no
rueg
icus
X-r
ay
2.6
Bos
taur
us
NM
R
n/a
Rat
tus
noru
egic
us
NM
R
n/a
Rat
tus
noru
egic
us
O'B
rien,
M.C
., F
lahe
rty,
K.M
., M
cKay
, D.B
. Lys
ine
71 o
f th
e ch
aper
one
prot
ein
Hsc
70 Is
ess
entia
l for
ATP
hy
drol
ysis
. J.B
iol.C
hem
. 19
96. v
271
p158
74
O'B
rien,
M.C
., F
lahe
rty,
K.M
., M
cKay
, D.B
. Lys
ine
71 o
f th
e ch
aper
one
prot
ein
Hsc
70 Is
ess
entia
l for
ATP
hy
drol
ysis
. J.B
iol.C
hem
. 19
96. v
271
p158
74
Cho
u, C
.C.,
et a
l. C
ryst
al s
truc
ture
of t
he C
-ter
min
al 1
0-
kDa
subd
omai
n of
Hsc
70. J
.Bio
l.Che
m.
2003
. v27
8 11
3031
1
Jian
g, J
., et
at.
Str
uctu
ral b
asis
of
inte
rdom
ain
com
mun
icat
ion
in th
e H
sc70
cha
pero
ne.
Mol
.Cel
l 200
5.
v20
p513
Mor
shau
ser,
R.C
., et
al.
Hig
h-re
solu
tion
solu
tion
stru
ctur
e of
the
18 k
Da
subs
trat
e-bi
ndin
g do
mai
n of
the
mam
mal
ian
chap
eron
e pr
otei
n H
sc70
. J.M
ol.B
iol.
1999
. v2
89 p
1387
Mor
shau
ser,
R.C
., et
al.
Hig
h-re
solu
tion
solu
tion
stru
ctur
e of
the
18
kD
a su
bstr
ate-
bind
ing
dom
ain
of th
e m
amm
alia
n ch
aper
one
prot
ein
Hsc
70. J
.Mol
.Bio
l. 19
99.
v289
p13
87
1 uoo
2H
RF
2H
RN
1 G4
l
I lM
2
1 DO
0
1 DO
2
1 HQ
Y
1 HT
1
Hsc
A (
subs
trat
e bi
ndin
g do
mai
n) -
lscU
pe
ptid
e co
mpl
ex
prot
ein
fold
ing,
hs
p70-
like
X-r
ay
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Esc
heric
hia
coli
Cup
p-V
icke
ry,
J.R
., et
al.
Cry
stal
Str
uctu
re o
f the
M
olec
ular
Cha
pero
ne H
scA
Sub
stra
te B
indi
ng D
omai
n C
ompl
exed
with
the
lsc
U R
ecog
nitio
n P
eptid
e E
LPP
VK
IHC
. J.M
ol.B
iol.
2004
. v34
2 p1
265
HS
co
l (m
utan
t) -
Cu'
m
eta
l ion
tr
ansp
ort
Ho
mo
sap
iens
B
anci
, L.
, et
al.
Hum
an S
co
l fun
ctio
nal
stud
ies
and
path
olog
ical
impl
icat
ions
of t
he P
174L
mut
ant.
P
roc.
Nat
l.Aca
d.S
ci.U
sa 2
007.
v10
4 p1
5
HS
col
(mut
ant)
- C
u'
me
tal i
on
tran
spor
t H
om
o s
apie
ns
Ban
ci,
L.,
et a
l. H
uman
Sc
ol f
unct
iona
l st
udie
s an
d pa
thol
ogic
al im
plic
atio
ns o
f the
P17
4L m
utan
t.
Pro
c.N
atl.A
cad.
Sci
.Usa
200
7. v
104
p15
Hsl
U
prot
ein
degr
adat
ion
(in
com
plex
with
H
slV
)
Hae
mop
hilu
s in
fluen
zae
Tra
me,
C.B
., M
cKay
, D
.B. S
truc
ture
of
Hae
mop
hilu
s in
fluen
zae
Hsl
U P
rote
in in
Cry
stal
s w
ith O
ne-d
imen
sion
al
Dis
orde
r T
win
ning
. A
cta
Cry
stal
logr
., S
ect.
D 2
001.
v57
p
lO7
9
Hsl
U
prot
ein
degr
adat
ion
(in
com
plex
with
H
slV
)
Hae
mop
hilu
s in
fluen
zae
Tra
me,
C.B
., M
cKay
, D.B
. S
truc
ture
of
Hae
mop
hilu
s in
fluen
zae
Hsl
U p
rote
in in
cry
stal
s w
ith o
ne-d
imen
sion
al
diso
rder
tw
inni
ng.
Act
a C
ryst
allo
gr.,
Sec
t.D
200
1. v
57
p107
9
Boc
htle
r, M
., et
al.
The
str
uctu
res
of
Hsl
U a
nd th
e A
TP
- de
pend
ent
prot
ease
Hsl
U-H
slV
. Nat
ure
2000
. v40
3 p
80
0
Hsl
U -
AD
P
Esc
heri
chia
co
li pr
otei
n de
grad
atio
n (in
co
mpl
ex w
ith
Hsl
V)
Hsl
U -
AN
P
prot
ein
degr
adat
ion
(in
com
plex
with
H
slV
)
Esc
heri
chia
co
li B
ocht
ler,
M.,
et a
l. T
he s
truc
ture
s of
Hsl
U a
nd th
e A
TP
- de
pend
ent
prot
ease
Hsl
U-H
slV
. N
atur
e 20
00. v
403
p800
Hsl
U -
Hsl
V -
AD
P
com
plex
pr
otei
n de
grad
atio
n E
sche
rich
ia c
oli
Wan
g, J
., et
al.
Nuc
leot
ide-
depe
nden
t co
nfor
mat
iona
l ch
ange
s in
a p
rote
ase-
asso
ciat
ed A
TP
ase
Hsl
U.
Str
uctu
re 2
001.
v9
p110
7
Hsl
U -
Hsl
V -
AD
P
com
ple
x pr
otei
n de
grad
atio
n E
sche
richi
a co
li W
ang,
J.,
et a
l. N
ucle
otid
e-de
pend
ent
conf
orm
atio
nal
chan
ges
in a
pro
teas
e-as
soci
ated
AT
Pas
e H
slU
. S
truc
ture
200
1. v
9 p1
107
Hsl
U -
Hsl
V -
AD
P
com
ple
x
Hsl
U -
Hsl
V -
AD
P
com
ple
x
Hsl
U -
Hsl
V -
Vin
yl
Su
lfon
e I
nhib
itor
Co
mp
lex
Hsl
U -
Hsl
V c
ompl
ex
Hsl
U -
Hsl
V c
ompl
ex
Hsl
U -
Hsl
V c
ompl
ex
Hsl
U -
Hsl
V c
ompl
ex
Hsl
U (
I-do
mai
n de
lete
d) -
Hsl
V -
AD
P
com
ple
x
Hsl
U (I
-dom
ain
de
lete
d)
- H
slV
- A
DP
co
mp
lex
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
prot
ein
degr
adat
ion
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
2.8
Esc
heric
hia
coli
4.1
6 B
acill
us s
ubtil
is
3.1
H
aem
ophi
lus
influ
enza
e R
d
2.8
Esc
heric
hia
coli
3.41
H
aem
ophi
lus
influ
enza
e
3.0
Esc
heric
hia
coli
7.0
Esc
heric
hia
coli
3.2
H
aem
ophi
lus
influ
enza
e
Wan
g, J
., et
al.
Nuc
leot
ide-
depe
nden
t co
nfor
mat
iona
l ch
ange
s in
a p
rote
ase-
asso
ciat
ed A
TP
ase
Hsl
U.
Str
uctu
re 2
001.
v9
p11
07
Wan
g, J
., et
al.
Cor
rect
ion
of X
-ray
int
ensi
ties
fro
m a
n H
slV
-Hsl
U c
o-cr
ysta
l co
ntai
ning
lat
tice-
tran
sloc
atio
n de
fect
s. A
cta
Cry
stal
logr
., S
ect.
D 2
005.
v61
p93
2
Sou
sa,
M.C
., et
al.
Cry
stal
Str
uctu
re o
f H
slU
V
Com
plex
ed w
ith a
Vm
yl S
ulfo
ne 1
nhib
itor:
Cor
robo
ratio
n of
a P
ropo
sed
Mec
hani
sm o
f A
llost
eric
Act
ivat
ion
of
Hsl
V
by
Hsl
U. J
.Mol
.Bio
l. 20
02.
v318
p77
9
Son
g, H
.K.,
et a
l. M
utat
iona
l S
tudi
es o
n H
slu
and
its
Doc
king
Mo
de
with
Hsl
v. P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
0.
v97
pl4
lO3
Sou
sa,
M.C
., e
t al
. C
ryst
al a
nd s
olut
ion
stru
ctur
es o
f an
H
slU
V p
rote
ase-
chap
eron
e co
mpl
ex.
Cel
l 200
0. v
103
p633
Wan
g, J
., et
al.
Cry
stal
str
uctu
res
of th
e H
slV
U
pept
idas
e-A
TP
ase
com
plex
rev
eal a
n A
TP
-dep
ende
nt
prot
eoly
sis
mec
hani
sm.
Str
uctu
re 2
001.
v9
p1
77
Wan
g, J
., et
al.
Cry
stal
str
uctu
res
of t
he H
slV
U
pept
idas
e-A
TP
ase
com
plex
rev
eal a
n A
TP
-dep
ende
nt
prot
eoly
sis
mec
hani
sm.
Str
uctu
re 2
001.
v9
p1
77
Kw
on, A
.R.,
et a
l. S
truc
ture
and
Rea
ctiv
ity o
f an
A
sym
met
ric
Com
plex
bet
wee
n H
slV
and
I-D
omai
n D
elet
ed H
slU
, a
Pro
kary
otic
Hom
olog
of
the
Euk
aryo
tic
Pro
teas
ome.
J.M
ol.B
iol.
2003
. v3
30 p
185
Kw
on,
A.R
., et
al.
Str
uctu
re a
nd R
eact
ivity
of
an
A
sym
met
ric C
ompl
ex b
etw
een
Hsl
V a
nd
I-D
omai
n D
elet
ed H
slU
, a P
roka
ryot
ic H
omol
og o
f the
Euk
aryo
tic
Pro
teas
ome.
J.M
ol.B
iol.
2003
. v3
30 p
185
2H53
1 IZ
Y
1 IZ
Z
1 N57
1 PV
2
1 HW
7
1 VQ
O
1 VZY
Hsp
16.3
(A
crl)
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
Cry
o-
EM
Cry
o-
EM
Cry
o-
EM
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Trit
icum
ae
stiv
um
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
The
rmot
oga
mar
itim
a
Bac
illus
sub
tilis
Ken
naw
ay,
C.K
., et
al.
Dod
ecam
eric
Str
uctu
re o
f the
S
mal
l Hea
t Sho
ck P
rote
in A
crl
from
Myc
obac
teriu
m
Tub
ercu
losi
s. T
o b
e p
ublis
hed.
Whi
te,
H.E
., e
t al
. M
ultip
le d
istin
ct a
ssem
blie
s re
veal
co
nfor
mat
iona
l fle
xibi
lity
in th
e sm
all h
eat s
hock
pro
tein
hs
p26.
Str
uctu
re 2
006.
v14
p11
97
Whi
te,
H.E
., et
al.
Mul
tiple
dis
tinct
ass
embl
ies
reve
al
conf
orm
atio
nal f
lexi
bilit
y in
the
smal
l hea
t sho
ck p
rote
in
hsp2
6. S
truc
ture
200
6. v
14 p
1197
Lee,
S.J
., et
al.
Cry
stal
str
uctu
res
of h
uman
DJ-
1 an
d E
sche
richi
a co
li H
sp31
, whi
ch s
hare
an
evol
utio
naril
y co
nser
ved
dom
ain.
J.B
iol.C
hem
. 200
3. v
278
p445
52
Lee,
S.J
., et
al.
Cry
stal
str
uctu
res
of h
uman
DJ-
1 an
d E
sche
richi
a co
li H
sp31
, whi
ch s
hare
an
evol
utio
naril
y co
nser
ved
dom
ain.
J.B
iol.C
hem
. 20
03.
v278
p44
552
Qui
gley
, P
.M.,
et a
l. T
he 1
.6A
Cry
stal
Str
uctu
re o
f the
C
lass
of C
hape
rone
Rep
rese
nted
by
Esc
heric
hia
coli
Hsp
31 R
evea
ls a
Put
ativ
e C
atal
ytic
Tria
d.
Pro
c.N
atl.A
cad.
Sci
.US
A 2
003.
vl 0
0 ~
31
37
Qui
gley
, P
.M.,
et a
l. A
new
nat
ive
EcH
sp31
str
uctu
re
sugg
ests
a k
ey r
ole
of s
truc
tura
l fle
xibi
lity
for
chap
eron
e fu
nctio
n. P
rote
in S
ci. 2
004.
v13
p26
9
Vija
yala
kshm
i, J.
, et
al.
The
2.2
A c
ryst
al s
truc
ture
of
Hsp
33: a
hea
t sho
ck p
rote
in w
ith r
edox
-reg
ulat
ed
chap
eron
e ac
tivity
. S
truc
ture
200
1. v
9 p3
67
Jaro
szew
ski,
L., e
t al.
Cry
stal
str
uctu
re o
f H
sp33
ch
aper
one
(TM
1394
) fr
om T
herm
otog
a m
ariti
ma
at 2
.20
A r
esol
utio
n. P
rote
ins
2005
. v61
p66
9
Jand
a, I
., et
al.
The
cry
stal
str
uctu
re o
f the
red
uced
, Z
n2+
-bou
nd f
orm
of t
he B
. sub
tilis
Hsp
33 c
hape
rone
and
its
impl
icat
ions
for
the
act
ivat
ion
mec
hani
sm.
Str
uctu
re
2004
. v12
p19
01
1 XJH
117F
2Q2G
1 HD
J
1 C3G
20
37
1 NLT
IXA
O
1 HJO
1 S3X
Hsp
33 9
N-t
erm
inal
do
mai
n)
Hsp
40
(dim
eriz
atio
n do
mai
n)
Hsp
40
(HD
J-1,
J-
dom
ain)
Hsp
40
(S
isl)
Hsp
4O (
Sis
l, J
- do
mai
n)
Hsp
40
(Y
djl)
- pe
ptid
e co
mpl
ex
Hsp
40
(Y
djl,
C-
term
inal
dom
ain)
Hsp
70
(A
TP
ase
dom
ain)
- A
DP
Hsp
70
(AT
Pas
e do
mai
n) -
AD
P
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp7O
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing
prot
ein
fold
ing
NM
R
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Esc
heric
hia
coli
Esc
heric
hia
coli
Cry
ptos
porid
ium
p
arv
um
Iow
a I1
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Hom
o sa
pien
s
Won
, H
.S.,
et a
l. T
he Z
inc-
depe
nden
t R
edox
Sw
itch
Dom
ain
of t
he C
hape
rone
Hsp
33 h
as a
Nov
el F
old.
J.
Mol
.Bio
l. 20
04. v
341
p893
Kim
, S.J
., et
al.
Cry
stal
str
uctu
re o
f pr
oteo
lytic
frag
men
ts
of th
e re
dox-
sens
itive
Hsp
33 w
ith c
onst
itutiv
e ch
aper
one
activ
ity.
Nat
.Str
uct.B
iol.
2001
. v8
p459
Wer
nim
ont,
A.K
., et
al.
Cry
stal
str
uctu
re o
f dim
eriz
atio
n do
mai
n of
HS
P40
from
Cry
ptos
porid
ium
par
vum
, cg
d2-1
800.
To
be
pub
lishe
d.
Qia
n, Y
.Q.,
et a
l. N
ucle
ar m
agne
tic r
eson
ance
sol
utio
n st
ruct
ure
of t
he h
uman
Hsp
40 (H
DJ-
1) J
-dom
ain.
J.
Mol
.Bio
l. 19
96. v
260
p224
Sha
, B
., C
yr,
D. P
urifi
catio
n, c
ryst
alliz
atio
n an
d pr
elim
inar
y X
-ray
cry
stal
logr
aphi
c st
udie
s of
S.
cere
visi
ae H
sp40
Sis
l. A
cta
Cry
stal
logr
., S
ect.D
199
9.
v55
pl2
34
Osi
piuk
, J.,
et a
l. X
-ray
cry
stal
str
uctu
re o
f J-d
omai
n of
S
isl
prot
ein,
Hsp
40 c
o-ch
aper
one
from
Sac
char
omyc
es
cere
visi
ae.
To
be
pub
lishe
d.
Li, J
., Q
ian,
X.,
Sha
, B
. T
he C
ryst
al S
truc
ture
of t
he
Yea
st H
sp40
Yd
jl C
ompl
exed
with
Its
Pep
tide
Sub
stra
te.
Str
uctu
re 2
003.
vl 1
p14
75
Wu,
Y.,
et a
l. T
he c
ryst
al s
truc
ture
of t
he C
-ter
min
al
frag
men
t of
yea
st H
sp40
Yd
jl re
veal
s no
vel d
imer
izat
ion
mot
if fo
r H
sp40
. J.M
ol.B
iol.
2005
. v34
6 p
l00
5
Osi
piuk
, J.
, e
t al.
Str
uctu
re o
f a n
ew c
ryst
al f
orm
of
hum
an H
sp70
AT
Pas
e do
mai
n. A
cta
Cry
stal
logr
., S
ect.D
19
99. v
55 p
1105
Spi
ram
, M
., et
al.
Hum
an H
sp70
mol
ecul
ar c
hape
rone
bi
nds
two
calc
ium
ions
with
in t
he A
TP
ase
dom
ain.
S
truc
ture
199
7. v
5 p4
03
1 BU
P
2P32
2826
1ZW
9
IZW
H
1 YE
R
IYE
S
1 US
U
Hsp
70 (
AT
Pas
e pr
otei
n fo
ldin
g do
mai
n, T
13S
mut
ant)
- A
DP
Hsp
70 (
C-t
erm
inal
10
prot
ein
fold
ing
kDa
subd
omai
n)
Hsp
7O (
Ssa
l , C
- pr
otei
n fo
ldin
g te
rmin
al d
omai
n) -
Hsp
40 (
Sis
l, C
- te
rmin
al d
omai
n)
com
plex
Hsp
82 -
inhi
bito
r pr
otei
n fo
ldin
g,
com
plex
ye
ast
hsp9
0
Hsp
82 -
rade
ster
pr
otei
n fo
ldin
g,
amin
e co
mpl
ex
yeas
t hs
p90
Hsp
9O
prot
ein
fold
ing
Hsp
9O
prot
ein
fold
ing
Hsp
9O -
Ah
al
com
plex
pr
otei
n fo
ldin
g
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
60
s ta
urus
Cae
norh
abdi
tis
eleg
ans
Dro
soph
ila
mel
anog
aste
r
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sou
sa,
M.C
., M
cKay
, D
.B. T
he h
ydro
xyl o
f thr
eoni
ne 1
3 of
the
bovi
ne 7
0-kD
a he
at s
hock
cog
nate
pro
tein
is
esse
ntia
l for
tran
sduc
ing
the
AT
P-in
duce
d co
nfor
mat
iona
l ch
ange
. B
ioch
emis
try
1998
. v37
p15
392
Wor
rall,
L.J
., W
alki
nsha
w,
M.D
. C
ryst
al s
truc
ture
of t
he
C-t
erm
inal
thre
e-he
lix b
undl
e su
bdom
ain
of C
. ele
gans
H
sp7O
. Bio
chem
.Bio
phys
.Res
.Com
mun
. 200
7. v
357
pi 0
5
Li, J
., W
u, Y
., Q
ian,
X.,
Sha
, B. C
ryst
al s
truc
ture
of y
east
S
isl
pept
ide-
bind
ing
frag
men
t an
d H
sp7O
Ssa
l C
- te
rmin
al c
ompl
ex.
Bi0
chem
.J. 2
006.
v39
8 p3
53
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T.,
Chi
osis
, G
. ln
hibi
tory
Li
gand
s A
dopt
Diff
eren
t Con
form
atio
ns W
hen
Bou
nd to
H
sp9O
or
GR
P94
: lm
plic
atio
ns f
or P
aral
og-s
peci
fic D
rug
Des
ign.
To
be p
ublis
hed
. v p
Imm
orm
ino,
R.M
., B
lagg
, B.S
., G
ewirt
h, D
.T.
lnhi
bito
ry
Liga
nds
Ado
pt D
iffer
ent
Con
form
atio
ns W
hen
Bou
nd to
H
sp9O
or
GR
P94
: lm
plic
atio
ns fo
r P
aral
og-s
peci
fic D
rug
Des
ign.
To
be p
ublis
hed.
Ste
bbin
s, C
.E.,
et a
l. C
ryst
al s
truc
ture
of a
n H
sp9O
- ge
ldan
amyc
in c
ompl
ex:
targ
etin
g o
f a p
rote
in c
hape
rone
by
an
antit
umor
age
nt. C
ell 1
997.
v89
p23
9
Ste
bbin
s, C
.E.,
et a
l. C
ryst
al s
truc
ture
of a
n H
sp9O
- ge
ldan
amyc
in c
ompl
ex:
targ
etin
g of
a p
rote
in c
hape
rone
by
an
antit
umor
age
nt.
Cel
l 199
7. v
89 p
239
Mey
er,
P.,
et a
l. S
truc
tura
l Bas
is fo
r R
ecru
itmen
t of
the
AT
Pas
e A
ctiv
ator
Ah
al
to th
e H
sp9O
Cha
pero
ne
Mac
hine
ry.
EM
BO
J.
2004
. v23
p14
02
1 US
V
IYE
T
2CG
9
2CG
E
1 US
7
2 F
XS
1 HK
7
2AK
P
1 AH
6
1 AH
8
Hsp
9O -
Ah
al
com
plex
Hsp
9O -
geld
anam
ycin
co
mpl
ex
Hsp
9O -
p23
(Sb
al)
co
mpl
ex
Hsp
90 -
p23
(S
ba
l)
com
plex
Hsp
9O -
p50
com
plex
Hsp
9O -
rad
amid
e co
mpl
ex
Hsp
9O (
mid
dle
dom
ain)
Hsp
9O (
mut
ant)
Hsp
9O (
N-t
erm
inal
do
mai
n)
Hsp
9O (
N-t
erm
inal
do
mai
n)
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Mey
er,
P.,
et a
l. S
truc
tura
l Bas
is fo
r R
ecru
itmen
t of t
he
AT
Pas
e A
ctiv
ator
Ah
al
to th
e H
sp9O
Cha
pero
ne
Mac
hine
ry. E
MB
O J
. 20
04. v
23 p
1402
Ste
bbin
s, C
.E.,
et a
l. C
ryst
al s
truc
ture
of
an H
sp9O
- ge
ldan
amyc
in c
ompl
ex: t
arge
ting
of a
pro
tein
cha
pero
ne
by
an a
ntitu
mor
age
nt. C
ell
1997
. v89
p23
9
Ali,
M.M
.U.,
et a
l. C
ryst
al S
truc
ture
of
an H
sp9O
- N
ucl
eo
tide
-P2
3lS
ba
l C
lose
d C
hape
rone
Com
plex
. N
atu
re 2
006.
v44
0 p1
013
Ali,
M.M
.U.,
et a
l. C
ryst
al S
truc
ture
of
an H
sp9O
- N
ucl
eo
tide
-P2
3lS
ba
l C
lose
d C
hape
rone
Com
plex
. N
atu
re 2
006.
v44
0 p1
013
Roe
, S
.M.,
et a
l. T
he M
echa
nism
of
Hsp
9O R
egul
atio
n b
y th
e P
rote
in K
inas
e-S
peci
fic C
ocha
pero
ne p
50(C
dc37
).
Cel
l 200
4. v
116
p87
Imm
orm
ino,
R.M
., G
ewirt
h, D
.T.,
Bla
gg, B
.S.
Inhi
bitto
ry
Liga
nds
Ado
pt D
iffer
ent C
onfo
rmat
ions
Whe
n B
ound
to
Hsp
9O o
r G
RP
94:
Impl
icat
ions
for
Par
alog
-spe
cific
Dru
g D
esig
n. T
o b
e p
ublis
hed.
Mey
er,
P.,
et a
l. S
truc
tura
l and
Fun
ctio
nal A
naly
sis
of t
he
M
iddl
e S
egm
ent o
f H
sp9O
. Im
plic
atio
ns fo
r A
TP
H
ydro
lysi
s an
d C
lient
Pro
tein
and
Coc
hape
rone
In
tera
ctio
ns. M
ol. C
ell 2
003.
vl I p
647
Ric
hter
, K
., et
al.
Intr
insi
c in
hibi
tion
of t
he
Hsp
9O A
TP
ase
activ
ity. J
.Bio
l.Che
m.
2006
. v2
81 p
1130
1
Pro
drom
ou, C
., et
al.
A m
olec
ular
cla
mp
in th
e c
ryst
al
stru
ctur
e of
the
N-t
erm
inal
dom
ain
of t
he
yea
st H
sp9O
ch
aper
one.
Nat
.Str
uct.B
iol.
1997
. v4
p477
Pro
drom
ou, C
., et
al.
A m
olec
ular
cla
mp
in th
e c
ryst
al
stru
ctur
e of
th
e N
-ter
min
al d
omai
n of
th
e y
east
Hsp
9O
chap
eron
e. N
at.S
truc
t.Bio
l. 19
97. v
4 p4
77
1 AM
W
1 BY
Q
1AM
1
1 A4H
2BR
C
2BR
E
2CG
F
1 BG
Q
1YC
1
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AD
P
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AD
P -
Mg
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AT
P
Hsp
9O (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
com
plex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l an
alog
ue c
ompl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l co
mpl
ex
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Pro
drom
ou, C
., et
al.
lden
tific
atio
n an
d st
ruct
ural
ch
arac
teriz
atio
n of
the
AT
PIA
DP
-bin
ding
site
in th
e H
sp9O
mol
ecul
ar c
hape
rone
. C
ell 1
997.
v90
p65
Obe
rman
n, W
.M.,
et a
l. In
viv
o fu
nctio
n of
Hsp
9O is
de
pend
ent o
n A
TP
bin
ding
and
AT
P h
ydro
lysi
s.
J.C
ell.B
iol.
1998
. v14
3 p9
01
Pro
drom
ou, C
., et
al.
Iden
tific
atio
n an
d st
ruct
ural
ch
arac
teriz
atio
n of
the
AT
PIA
DP
-bin
ding
site
in th
e H
sp9O
mol
ecul
ar c
hape
rone
Cel
l 19
97. v
90 p
65
Pro
drom
ou, C
., et
al.
lden
tific
atio
n an
d st
ruct
ural
ch
arac
teriz
atio
n of
the
AT
PIA
DP
-bin
ding
site
in th
e H
sp9O
mol
ecul
ar c
hape
rone
. C
ell
1997
. v90
p65
Che
ung,
K.-
M.J
., et
al.
The
Id
en
tif~
catio
n, S
ynth
esis
, P
rote
in C
ryst
al S
truc
ture
and
in
Vitr
o B
ioch
emic
al
Eva
luat
ion
of a
New
3,4
-Dia
rylp
yraz
ole
Cla
ss o
f H
sp9O
In
hibi
tors
. Bio
org.
Med
.Che
m.L
ett.
2005
. v1
5 p
3338
Che
ung,
K.-
M.J
., et
al.
The
Ide
ntifi
catio
n, S
ynth
esis
, P
rote
in C
ryst
al S
truc
ture
and
in V
itro
Bio
chem
ical
E
valu
atio
n of
a N
ew 3
,4-D
iary
lpyr
azol
e C
lass
of
Hsp
9O
Inhi
bito
rs. B
ioor
g.M
ed.C
hem
.Let
t. 20
05. v
15 p
3338
Pro
isy,
N.,
et a
l. In
hibi
tion
of H
sp9O
with
Syn
thet
ic
Mac
rola
cton
es: S
ynth
esis
and
Str
uctu
ral a
nd B
iolo
gica
l E
valu
atio
n of
Rin
g a
nd C
onfo
rmat
iona
l Ana
logs
of
Rad
icic
ol. C
hem
.Bio
l. 20
06. v
13 p
1203
Roe
, S.M
., et
al.
Str
uctu
ral b
asis
for
inhi
bitio
n of
the
H
sp9O
mol
ecul
ar c
hape
rone
by
the
antit
umor
ant
ibio
tics
radi
cico
l and
gel
dana
myc
in. J
.Med
.Che
m.
1999
. v42
p2
60
Kre
usch
, A.,
et a
l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
g.M
ed.C
hem
.Let
t. 20
05. v
15 p
1475
1YC
3
1 YC
4
2U
WD
1 UY
L
1 UY
G
IUY
I
IUY
F
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
Hsp
9Oa
- inh
ibito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n)
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(
2,5-
di
met
hoxy
-ben
zyl)-
2-
fluor
o-9H
-pur
in-6
- yl
amin
e co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(
2,5-
di
met
hoxy
-ben
zyl)
-2-
fluor
o-9-
pent
-9H
-pur
in-
6-yl
amin
e co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(2
-chl
oro-
3,
4,5-
trim
etho
xy-
benz
yl)-
2-flu
oro-
9-
pent
-4-y
lnyl
-9H
-pur
in-
6-yl
amin
e co
mpl
ex
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Kre
usch
, A.,
et a
l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
g.M
ed.C
hem
.Let
t. 20
05.
v15
p147
5
Kre
usch
, A,,
et a
l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
g.M
ed.C
hem
.Let
t. 20
05. v
15
p14
75
Sha
rp,
S.Y
., et
al.
Inhi
bitio
n of
the
Hea
t S
hock
Pro
tein
90
M
olec
ular
Cha
pero
ne in
Vitr
o a
nd
in V
ivo
by
Nov
el,
Syn
thet
ic,
Pot
ent
Res
orci
nylic
Pyr
azol
e/ls
oxaz
ole
Am
ide
Ana
logu
es.
Mol
.Can
cer T
her.
200
7. v
6 p1
198
Wrig
ht,
L.,
et a
l. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
m.B
iol.
2004
. v
l I p7
75
Wrig
ht,
L.,
et a
l. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
m.B
iol.
2004
. v
l I p7
75
Wrig
ht,
L., e
t al
. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
m.B
iol.
2004
. v
l I p7
75
Wrig
ht,
L., e
t al
. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
.Bio
l. 20
04.
vl I p
775
1 UY
6 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9
-but
yl-8
- (3
,4,5
-trim
etho
xy-
benz
yl)-
9H-p
urin
-6-
ylam
ine
com
plex
1 UY
8 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9-
buty
l-8-(
3-
met
hoxy
-ben
zy1)
-9H
- pu
rin-6
-yla
min
e co
mpl
ex
1 UY
7 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9
-but
yl-8
-(4-
m
etho
xy-b
enzy
l)-9H
- pu
rin-6
-yla
min
e co
mpl
ex
2BS
M
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
2BT
0 H
sp9O
a (N
-ter
min
al
dom
ain)
- in
hibi
tor
com
plex
2BY
H
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
2BY
I H
sp9O
a (N
-ter
min
al
dom
ain)
- in
hibi
tor
com
plex
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s W
right
, L.
, et
al.
Str
uctu
re-A
ctiv
ity R
elat
ions
hips
in
Pur
ine-
Bas
ed ln
hibi
tor
Bin
ding
to H
sp9O
Isof
orm
s.
Che
m.B
io1.
200
4. v
l I p7
75
prot
ein
fold
ing
X-r
ay
1.98
H
omo
sapi
ens
Wrig
ht,
L., e
t al
. Str
uctu
re-A
ctiv
ity R
elat
ions
hips
in
Pur
ine-
Bas
ed ln
hibi
tor
Bin
ding
to H
sp9O
Isof
orm
s.
Che
m.B
iol.
2004
. v
l I p7
75
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s W
right
, L.
, et
al. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
.Bio
l. 20
04.
vl I p7
75
prot
ein
fold
ing
X-r
ay
2.05
H
omo
sapi
ens
Dym
ock,
B.W
., et
al.
Nov
el, P
oten
t Sm
all-M
olec
ule
lnhi
bito
rs o
f the
Mol
ecul
ar C
hape
rone
Hsp
9O D
isco
vere
d T
hrou
gh S
truc
ture
-Bas
ed D
esig
n. J
.Med
.Che
m.
2005
. v4
8 p4
212
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s D
ymoc
k, B
.W.,
et a
l. N
ovel
, P
oten
t Sm
all-M
olec
ule
lnhi
bito
rs o
f the
Mol
ecul
ar C
hape
rone
Hsp
9O D
isco
vere
d T
hrou
gh S
truc
ture
-Bas
ed D
esig
n. J
.Med
.Che
m.
2005
. v4
8 p4
2 12
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s B
roug
h, P
.A.,
et a
l. 3-
(5-C
hlor
o-2,
4-D
ihyd
roxy
phen
y1)-
P
yraz
ole-
4-C
arbo
xam
ides
as
lnhi
bito
rs o
f the
Hsp
9O
Mol
ecul
ar C
hape
rone
. Bio
org.
Med
.Che
m.L
ett.
2005
. v15
p5
197
prot
ein
fold
ing
X-r
ay
1.6
Hom
o sa
pien
s B
roug
h, P
.A.,
et a
l. 3-
(5-C
hlor
o-2,
4-D
ihyd
roxy
phen
y1)-
P
yraz
ole-
4-C
arbo
xam
ides
as
lnhi
bito
rs o
f the
Hsp
9O
Mol
ecul
ar C
hape
rone
. B
ioor
g.M
ed.C
hem
.Let
t. 20
05.
v15
p519
7
2H55
2F
Wz
2FW
Y
1 UY
M
1 XQ
R
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-DZ
8 in
hibi
tor
com
plex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-H64
in
hibi
tor
com
plex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-H71
in
hibi
tor
com
plex
Hsp
9OP
- 9-
buty
l- 8(
3,4,
5-tr
imet
hoxy
- be
nzyl
)-9H
-pur
in-6
- yl
amin
e
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
Hsp
BP
l (c
ore
dom
ain)
re
gula
tion
of
Hsp
70 fu
nctio
n
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
1.9
Hom
o sa
pien
s
1.79
H
omo
sapi
ens
2.3
Hom
o sa
pien
s
2.7
Hom
o sa
pien
s
1.9
Hom
o sa
pien
s
2.0
Hom
o sa
pien
s
2.1
Hom
o sa
pien
s
2.1
Hom
o sa
pien
s
2.45
H
omo
sapi
ens
2.1
Hom
o sa
pien
s
Bar
ril, X
., et
al.
Str
uctu
re-B
ased
Dis
cove
ry o
f a
New
C
lass
of H
sp9O
Inh
ibito
rs. B
ioor
g.M
ed.C
hem
.Let
t. 20
05.
vl5
~5
18
7
Bar
ril, X
., et
al.
4-A
min
o D
eriv
ativ
es o
f th
e H
sp9O
In
hibi
tor C
ct01
8159
. B
ioor
g.M
ed.C
hem
.Let
t. 20
06. v
16
p254
3
Bar
ril, X
., et
al.
4-A
min
o D
eriv
ativ
es o
f th
e H
sp9O
ln
hibi
tor
Cct
0181
59.
Bio
org.
Med
.Che
m.L
ett.
2006
. v1
6 p2
543
Bar
ril, X
., et
al.
4-A
min
o D
er~
vativ
es of t
he H
sp9O
ln
hibi
tor
Cct
0181
59.
Bio
org.
Med
.Che
m.L
ett.
2006
. v16
p2
543
How
es, R
., et
al.
A F
luor
esce
nce
Pol
ariz
atio
n A
ssay
for
Inhi
bito
rs o
f H
sp9O
. Ana
l.Bio
chem
. 200
6. v
350
p202
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
hem
ical
st
udie
s of
8-a
ryl-s
ulfa
nyl a
deni
ne c
lass
Hsp
9O in
hibi
tors
. J.
Med
.Che
m. 2
006.
v49
p49
53
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
he
m~
cal
stud
ies
of 8
-ary
l-sul
fany
l ade
nine
cla
ss H
sp9O
inhi
bito
rs.
J.M
ed.C
hem
. 200
6. v
49 p
4953
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
hem
ical
st
udie
s of
8-a
ryl-s
ulfa
nyl a
deni
ne c
lass
Hsp
9O in
hibi
tors
. J.
Med
.Che
m. 2
006.
v49
p49
53
Wrig
ht,
L., e
t al
. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
.Bio
l. 20
04. v
l 1 p
775
Sho
mur
a, Y
., et
al.
Reg
ulat
ion
of H
sp70
Fun
ctio
n by
H
spB
PI;
Str
uctu
ral A
naly
sis
Rev
eals
an
Alte
rnat
e M
echa
nism
for
Hsp
70 N
ucle
otid
e E
xcha
nge.
Mol
.Cel
l 20
05. v
17 p
367
1 XQ
S
21W
S
21W
U
21W
X
1 SF
8
210Q
1 Y
4S
1 Y
4U
210P
Hsp
BP
l (c
ore
dom
ain)
re
gula
tion
of
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Ho
mo
sap
iens
S
hom
ura,
Y.,
et a
l. R
egul
atio
n of
Hsp
70 F
unct
ion
by
Hsp
BP
l; S
truc
tura
l Ana
lysi
s R
evea
ls a
n A
ltern
ate
Mec
hani
sm fo
r H
sp70
Nuc
leot
ide
Exc
hang
e. M
ol.C
ell
2005
. v1
7 p3
67
- H
sp70
(A
TP
ase
dom
ain)
com
plex
~
g~
70
fu
nctio
n
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l pr
otei
n fo
ldin
g,
Sac
char
omyc
es
cere
visi
ae
Pro
isy,
N.,
et a
l. ln
hibi
tion
of H
sp9O
with
Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd S
truc
tura
l an
d B
iolo
gica
l E
valu
atio
n of
Rin
g an
d C
onfo
rmat
iona
l Ana
logs
of
Rad
icic
ol.
Che
m.B
iol.
2006
. v1
3 p1
203
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l pr
otei
n fo
ldin
g,
Sac
char
omyc
es
cere
visi
ae
Pro
isy,
N.,
et a
l. ln
hibi
tion
of H
sp9O
with
Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd S
truc
tura
l and
Bio
logi
cal
Eva
luat
ion
of R
ing
an
d C
onfo
rmat
iona
l Ana
logs
of
Rad
icic
ol.
Che
m.B
iol.
2006
. v1
3 p1
203
Pro
isy,
N.,
et a
l. ln
hibi
tion
of H
sp9O
with
Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd
Str
uctu
ral a
nd
Bio
logi
cal
Eva
luat
ion
of R
ing
and
Con
form
atio
nal A
nalo
gs o
f R
adic
icol
. C
hem
.Bio
l. 20
06.
v13
p120
3
Hsp
9O (
N-t
erm
inal
do
mai
n) -
rad
icic
ol
prot
ein
fold
ing,
S
acch
arom
yces
ce
revi
siae
Htp
G
prot
ein
fold
ing,
E
.co
li hs
p90
Esc
heric
hia
coli
Har
ris,
S.F
., S
hiau
, A.K
., A
gard
, D
.A. T
he C
ryst
al
Str
uctu
re o
f the
Car
boxy
-ter
min
al D
imer
izat
ion
Dom
ain
of h
tpG
, th
e E
sche
richi
a co
li H
sp90
, Rev
eals
a P
oten
tial
Sub
stra
te B
indi
ng S
ite.
Str
uctu
re 2
004.
v12
p10
86
Htp
G
prot
ein
fold
ing,
E. c
oli
hsp9
0 E
sche
richi
a co
li S
hiau
, A.K
., et
al.
Str
uctu
ral A
naly
sis
of E
. col
i hsp
90
reve
als
dram
atic
nuc
leot
ide-
depe
nden
t co
nfor
mat
iona
l re
arra
ngem
ents
. C
ell 2
006.
v12
7 p3
29
Htp
G -
AD
P
prot
ein
fold
ing,
E. c
oli
hsp9
0 E
sche
richi
a co
li H
uai,
Q.,
et a
l. C
onfo
rmat
ion
rear
rang
emen
t of
hea
t sh
ock
prot
ein
90 u
pon
AD
P b
indi
ng.
Str
uctu
re 2
005.
v13
p5
79
Htp
G -
AD
P
prot
ein
fold
ing,
E. c
oli
h s p
9O
Esc
heric
hia
coli
Hua
i, Q
., et
al.
Con
form
atio
n re
arra
ngem
ent
of h
eat
shoc
k pr
otei
n 90
upo
n A
DP
bin
ding
. S
truc
ture
200
5. v
13
p579
Htp
G -
AD
P
prot
ein
fold
ing,
E. c
oli
h sp
9O
Esc
heric
hia
coli
Shi
au, A
.K.,
et a
l. S
truc
tura
l Ana
lysi
s of
E. c
oli h
sp90
re
veal
s dr
amat
ic n
ucle
otid
e-de
pend
ent
conf
orm
atio
nal
rear
rang
emen
ts.
Cel
l 200
6. v
127
p329
Htp
G (
mid
dle
dom
ain)
pr
otei
n fo
ldin
g,
E. c
oli
hsp9
0 X
-ray
1.
9 E
sche
richi
a co
li S
hiau
, A.K
., et
al.
Str
uctu
ral A
na
lys~
s of
E. c
oli h
sp90
R
evea
ls D
ram
atic
Nuc
leot
ide-
Dep
ende
nt C
onfo
rmat
iona
l R
earr
ange
men
ts. C
ell 2
006.
v12
7 p3
29
Shi
au, A
.K.,
et a
l. S
truc
tura
l Ana
lysi
s of
E. c
oli h
sp90
re
veal
s dr
amat
ic n
ucle
otid
e-de
pend
ent c
onfo
rmat
iona
l re
arra
ngem
ents
. C
ell 2
006.
v12
7 p3
29
Toc
hio,
N.,
et a
l. T
he s
olu
t~o
n stru
ctur
e o
f the
C-t
erm
inal
do
mai
n of
hum
an A
ctiv
ator
of 9
0 kD
a he
at s
hock
pro
tein
A
TP
ase
hom
olog
1. T
o be
pub
lishe
d.
Htp
G (N
-ter
min
al
dom
ain)
- A
DP
pr
otei
n fo
ldin
g,
E. c
oli
hsp9
0 1.
65
Esc
heric
hia
coli
X-r
ay
Hum
an A
ctiv
ator
of
hsp9
0 A
TP
ase
hom
olog
1 (C
-ter
min
al
dom
ain)
nla
H
omo
sapi
ens
NM
R
Lam
b, A
.L.,
et a
l. C
ryst
al s
truc
ture
of t
he s
econ
d do
mai
n of
the
hum
an c
oppe
r ch
aper
one
for
supe
roxi
de
dism
utas
e. B
ioch
emis
try
2000
. v39
p15
89
hum
an c
oppe
r ch
aper
one
for
supe
roxi
de d
ism
utas
e (h
CC
S, d
omai
n II)
- zn
2+
Hum
an D
J-1
met
al io
n tr
ansp
ort
X-r
ay
2.75
H
omo
sapi
ens
nla
X
-ray
1.
2 H
omo
sapi
ens
Witt
, A.C
., La
kshm
inar
asim
han,
M.,
Wils
on,
M.A
. P
re-
oxid
atio
n C
ompl
ex o
f H
uman
DJ-
1. T
o b
e p
ublis
hed.
hum
an K
IM0
88
5
prot
ein
(firs
t col
d-
shoc
k do
mai
n)
regu
latio
n of
tr
ansc
riptio
n N
MR
n
la
Hom
o sa
pien
s G
oron
cy, A
., et
al.
Sol
utio
n st
ruct
ure
of th
e fir
st c
old-
sh
ock
dom
ain
of t
he h
uman
KIM
08
85
pro
tein
(U
NR
pr
otei
n). T
o b
e p
ublis
hed.
nla
Gor
oncy
, A,,
et a
l. S
olut
ion
stru
ctur
e of
the
third
col
d-
shoc
k do
mai
n of
the
hum
an K
IM0
88
5 p
rote
in (
UN
R
PR
OT
EIN
). T
o be
pub
lishe
d.
hum
an K
IM0
88
5
prot
ein
(thr
id c
old-
sh
ock
dom
ain)
lnvB
- S
ipA
com
plex
NM
R
nla
H
omo
sapi
ens
type
Ill
prot
ein
secr
etio
n X
-ray
2.
2 S
alm
onel
la
typh
imur
ium
Li
lic, M
., et
al.
A c
omm
on s
truc
tura
l mot
if in
the
bind
ing
of v
irule
nce
fact
ors
to b
acte
rial s
ecre
tion
chap
eron
es.
Mol
.Cel
l 200
6. v
21 p
653
Lipa
se c
hape
rone
(C
- te
rmin
al d
omai
n) -
lipas
e co
mpl
ex
type
II
secr
etio
n,
perip
lasm
ic
ster
ic
chap
eron
e
X-r
ay
1.85
B
urkh
olde
ria
glum
ae
Pau
wel
s, K
., et
al.
Str
uctu
re o
f a
mem
bran
e-ba
sed
ster
ic
chap
eron
e in
com
plex
with
its
lipas
e su
bstr
ate.
N
at.S
truc
t.Mol
.Bio
l. 20
06.
v13
p374
1 IW
L
1 UA
8
2CU
G
1 ZX
J
1 TR
8
Lol A
lip
opro
tein
tr
ansp
ort
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
1.65
E
sche
richi
a co
li T
aked
a, K
., et
. al.
Cry
stal
str
uctu
res
of b
acte
rial
lipop
rote
in lo
caliz
atio
n fa
ctor
s, L
olA
and
Lol
B.
EM
BO
J.
2003
. v2
2 p3
199
LolA
lip
opro
tein
tr
ansp
ort
Esc
heric
hia
coli
Tak
eda,
K.,
et. a
l. C
ryst
al s
truc
ture
s of
bac
teria
l lip
opro
tein
loca
lizat
ion
fact
ors,
Lol
A a
nd L
olB
. E
MB
O J
. 20
03.
v22
~3
19
9
mK
IAA
0962
(J-
dom
ain)
M
us m
uscu
lus
Ohn
ishi
, S.,
et a
l. S
olut
ion
stru
ctur
e of
the
J do
mai
n of
th
e ps
eudo
Dna
J pr
otei
n, m
ouse
hyp
othe
tical
m
KIA
A09
6. T
o b
e pu
blis
hed.
nla
Myc
opla
sma
pneu
mon
iae
Sch
ulze
-Gah
men
, U.,
et a
l. S
truc
ture
of t
he h
ypot
hetic
al
Myc
opla
sma
prot
ein
MP
N55
5 su
gges
ts a
cha
pero
ne
func
tion.
Act
a C
ryst
allo
gr.,
Sec
t.D 2
005.
v61
p13
43
Nas
cent
pol
ypep
tide-
as
soci
ated
com
plex
P
AC
)
prev
entio
n of
im
prop
er
prot
ein
asso
ciat
ions
Met
hano
ther
mo
bact
er
mar
burg
ensi
s
Spr
eter
, T.,
Pec
h, M
., B
eatr
ix, B
. The
cry
stal
str
uctu
re o
f ar
chae
al n
asce
nt p
olyp
eptid
e-as
soci
ated
com
plex
(N
AC
) re
veal
s a
uniq
ue fo
ld a
nd t
he p
rese
nce
of a
UB
A
dom
ain.
J.B
iol.C
hem
. 200
5. v
280
p158
49
1 XB
9
1 XEO
2P1
B
1 K
5J
1 N
LQ
hist
one
chap
eron
e in
th
e nu
cleo
lus
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Xen
opus
laev
is
Nam
bood
iri, V
.M.,
et a
l. T
he S
truc
ture
and
Fun
ctio
n of
X
enop
us N
03
8-C
ore
, a H
isto
ne C
hape
rone
in th
e N
ucle
olus
. Str
uctu
re 2
004.
v12
p21
49
hist
one
chap
eron
e in
th
e nu
cleo
lus
Xen
opus
laev
is
Nam
bood
iri, V
.M.,
et a
l. T
he S
truc
ture
and
Fun
ctio
n of
X
enop
us N
03
8-C
ore
, a H
isto
ne C
hape
rone
in th
e N
ucle
olus
. Str
uctu
re 2
004.
v12
p21
49
nucl
eoph
osm
in-c
ore
Hom
o sa
pien
s Le
e, H
.H.,
et a
l. C
ryst
al S
truc
ture
of
Hum
an
Nuc
leop
hosm
in-C
ore
Rev
eals
Pla
stic
ity o
f the
Pen
tam
er-
Pen
tam
er I
nter
face
. To
be P
ublis
hed.
nucl
eoso
me
asse
mbl
y
Nuc
leop
lasm
in c
ore
nucl
eoso
me
asse
mbl
y X
enop
us la
e vi
s D
utta
, S.,
et a
l. T
he c
ryst
al s
truc
ture
of
nucl
eopl
asm
in-
core
: im
plic
atio
ns fo
r hi
ston
e bi
ndin
g an
d nu
cleo
som
e as
sem
bly.
Mol
.Cel
l 200
1. v
8 p8
41
Nuc
leop
lasm
in-li
ke
prot
ein
(NLP
, N
- te
rmin
al c
ore)
hist
one
chap
eron
e D
roso
phila
m
elan
ogas
ter
Nam
bood
iri, V
.M.H
. et
al.
The
cry
stal
str
uctu
re o
f D
roso
phila
NLP
-cor
e P
rovi
des
Insi
ght i
nto
Pen
tam
er
For
mat
ion
and
His
tone
Bin
ding
. Str
uctu
re 2
003.
vl I
p175
2AY
U
1 EJF
2PJH
1 QP
P
1 QP
X
3DP
A
2J7L
1 NO
L
2J2Z
Nuc
leos
ome
asse
mbl
y pr
otei
n 1
prot
ein
asse
mbl
y
p97
N d
omai
n- n
p14
UB
D c
ompl
ex
Pap
D
Pap
D
Pap
D
Pap
D -
inhi
bito
r co
mpl
ex
Pap
D -
Pap
E (
N-
term
inal
-del
eted
) co
mpl
ex
Pap
D -
Pap
H c
ompl
ex
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp90
n /a
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Mu
s m
uscu
lus
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Esc
heric
hia
coli
Par
k, Y
.J.,
Luge
r, K
. The
str
uctu
re o
f nu
cleo
som
e as
sem
bly
prot
ein
1. P
roc.
Nat
l.Aca
d.S
ci.U
sa 2
006.
v10
3 p
l24
8
Wea
ver,
A.J
., et
al.
Cry
stal
str
uctu
re a
nd a
ctiv
rty
of
hum
an p
23, a
hea
t sho
ck p
rote
in 9
0 co
-cha
pero
ne.
J.B
iol.C
hem
. 20
00. v
275
p230
45
Issa
cson
, R
., et
al.
Det
aile
d st
ruct
ural
ins
ight
s in
to th
e p9
7-N
p14-
Ufd
l in
terf
ace.
To
be p
ublis
hed.
Hun
g, D
.L.,
et a
l. S
truc
tura
l bas
is o
f ch
aper
one
self-
ca
ppin
g in
P p
ilus
biog
enes
is.
Pro
c.N
atl.A
cad.
Sci
.US
A
1999
. v96
p81
78
Hun
g, D
.L.,
et a
l. S
truc
tura
l bas
is o
f ch
aper
one
self-
ca
ppin
g in
P p
ilus
biog
enes
is.
Pro
c.N
atl.A
cad.
Sci
.US
A
1999
. v96
pa
l78
Hol
mgr
en, A
., B
rand
en,
C.I.
Cry
stal
str
uctu
re o
f ch
aper
one
prot
ein
Pap
D r
evea
ls a
n im
mun
oglo
bulin
fol
d.
Nat
ure
1989
. v34
2 p2
48
Pin
kner
, J.S
., et
al.
Rat
iona
lly D
esig
ned
Sm
all
Com
poun
ds I
nhib
it P
ilus
Bio
gene
sis
in U
ropa
thog
enic
B
acte
ria.
Pro
c.N
atl.A
cad.
Sci
.US
A 2
006.
v10
3 p1
7897
Sau
er,
F.G
., et
al.
Cha
pero
ne p
rimin
g of
pilu
s su
buni
ts
faci
litat
es a
topo
logi
cal
tran
sitio
n th
at d
rives
fibe
r fo
rmat
ion.
Cel
l 200
2. v
l 1 I p5
43
Ver
ger,
D.,
et a
l. M
olec
ular
Mec
hani
sm o
f P
Pilu
s T
erm
inat
ion
in U
ropa
thog
enic
Esc
heric
hia
Col
i. E
MB
O
Rep
orts
200
6. v
7 p
1228
1 PD
K
1 Y6Z
1 IT
P
1 FX
K
1 ALO
1 CD
3
2UW
J
2GZ
P
2FC
W
Pa
pD
- P
apK
com
plex
ce
ll w
all
orga
niza
tion
and
biog
enes
is
n/a
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
NM
R
X-r
ay
Esc
heri
chia
co
li S
auer
, F
.G.,
et a
l. S
truc
tura
l bas
is o
f ch
aper
one
func
tion
and
pilu
s bi
ogen
esis
. S
cien
ce 1
999.
v28
5 ~
10
58
PF
14-0
417
prot
ein
(C-
term
inal
dom
ain)
P
lasm
od
ium
fa
lcip
arum
30
7
Ved
adi,
M.,
et a
l. G
enom
e-sc
ale
prot
ein
expr
essi
on a
nd
stru
ctur
al b
iolo
gy o
f P
lasm
odiu
m f
alci
paru
m a
nd r
elat
ed
Api
com
plex
an o
rgan
ism
s. M
ol.B
ioch
em.P
aras
ito1.
200
7.
v151
pl0
0
intr
amol
ecul
ar
chap
eron
e P
leur
otus
os
trea
tus
Sas
akaw
a, H
., et
al.
Str
uctu
re o
f P
OIA
I, a
hom
olog
ous
prot
ein
to t
he p
rope
ptid
e of
sub
tilis
in:
impl
icat
ion
for
prot
ein
fold
abili
ty a
nd t
he fu
nctio
n a
s an
intr
amol
ecul
ar
chap
eron
e. J
.Mol
.Bio
l. 20
02. v
317
p159
Sie
gert
, R
., et
al.
Str
uctu
re o
f th
e m
olec
ular
cha
pero
ne
pref
oldi
n: u
niqu
e in
tera
ctio
n o
f m
ultip
le c
oile
d co
il te
ntac
les
with
unf
olde
d pr
otei
ns.
Cel
l 200
0. v
103
p621
prot
ein
fold
ing
Pre
fold
in (
Gim
C)
Met
hano
bact
eri
um
th
erm
oaut
otro
p h
icu
m
proc
apsi
d vi
ral p
roca
psid
m
atur
atio
n B
acte
riop
hage
P
HI-
XI 7
4 D
okla
nd,
T.,
et a
l. S
truc
ture
of
a vi
ral p
roca
psid
with
m
olec
ular
sca
ffold
ing.
Nat
ure
1997
. v38
9 p3
08
Pro
caps
id
vira
l pro
caps
id
mat
urat
ion
Bac
teri
opha
ge
PH
I-X
I 75
Dok
land
, T
., et
al.
The
rol
e o
f sc
affo
ldin
g pr
otei
ns in
the
asse
mbl
y o
f th
e s
mal
l, si
ngle
-str
ande
d D
NA
vir
us
phiX
174.
J.M
ol.B
iol.
1999
. v28
8 p5
95
Psc
E - P
scG
- P
scF
co
mpl
ex
type
Ill
prot
ein
secr
etio
n P
seud
omon
as
aeru
gino
sa
Qui
naud
, M
., et
al.
Str
uctu
re o
f th
e H
eter
otri
mer
ic
Com
plex
tha
t R
egul
ates
Typ
e Il
l Sec
retio
n N
eedl
e F
orm
atio
n. P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
7. v
104
p780
3
Sal
mon
ella
ty
phim
uriu
m
Par
ish,
D.,
et a
l. S
olut
ion
NM
R s
truc
ture
of Q
8ZP
24 fr
om
Sal
mon
ella
typh
imur
ium
LT
2. T
o b
e p
ublis
hed.
Fis
her,
C.,
Beg
lova
, N.,
Bla
cklo
w, S
.C.
Str
uctu
re o
f an
LDLR
-RA
P C
ompl
ex R
evea
ls a
Gen
eral
Mo
de
for
Liga
nd
Rec
ogni
tion
by
Lipo
prot
ein
Rec
epto
rs.
Mol
. C
ell 2
006.
v2
2 p2
77
RA
P -
LDLR
com
plex
a
spec
ializ
ed
chap
eron
e fo
r en
docy
tic
rece
ptor
s
Ho
mo
sap
iens
2CQ
Q
RS
Gl R
UH
-037
2CQ
R
RS
Gl R
UH
-038
2C
06
S
afB
- S
afA
com
plex
2C
07
S
afB
- S
afA
com
plex
IWP
O
SC
Ol
2GT
5 S
col
(C-t
erm
inal
do
mai
n)
2GV
P
Sco
l (C
-ter
min
al
dom
ain)
2GQ
M
Sco
l (C
-ter
min
al
dom
ain)
- C
u'
tubu
lin f
oldi
ng,
hom
olog
of
beta
-tub
ulin
sp
ecifi
c co
fact
or A
n/a
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
prot
ein
asse
mbl
y,
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
X-r
ay
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
NM
R
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Hom
o sa
pien
s
Sal
mon
ella
ty
phim
uriu
m
Sal
mon
ella
ty
phim
uriu
m
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Ste
inba
cher
, S.
Cry
stal
str
uctu
re o
f the
pos
t-ch
aper
onin
be
ta-t
ubul
in b
indi
ng c
ofac
tor
Rbl
2p. N
at.S
truc
t.Bio
l. 19
99. v
6 p1
029
Doi
-Kat
ayam
a, Y
., et
al.
Sol
utio
n S
truc
ture
of
RS
Gl
RU
H-0
37, a
myb
DN
A-b
indi
ng d
omai
n in
hum
an c
DN
A.
To
be
pub
lishe
d.
Doi
-Kat
ayam
a, Y
., e
t al.
Sol
utio
n st
ruct
ure
of R
SG
l RU
H-
043,
a m
yb D
NA
-bin
ding
dom
ain
in h
uman
cD
NA
. To
be
publ
ishe
d.
Rem
aut,
H.,
et a
l. D
onor
-Str
and
Exc
hang
e in
C
hape
rone
-Ass
iste
d P
ilus
Ass
embl
y P
roce
eds
Thr
ough
a
Con
cert
ed B
eta-
Str
and
Dis
plac
emen
t Mec
hani
sm.
Mol
ecul
ar C
ell 2
006.
v22
p83
1
Rem
aut,
H.,
et a
l. D
onor
-Str
and
Exc
hang
e in
C
hape
rone
-Ass
iste
d P
ilus
Ass
embl
y P
roce
eds
Thr
ough
a
Con
cert
ed B
eta-
Str
and
Dis
plac
emen
t Mec
hani
sm.
Mol
ecul
ar C
ell 2
006.
v22
p83
1
Will
iam
s, J
.C.,
et a
l. C
ryst
al S
truc
ture
of
Hum
an S
CO
1:
impl
icat
ions
for
redo
x si
gnal
ing
by a
mito
chon
dria
1 cy
toch
rom
e c
oxid
ase
"ass
embl
y" p
rote
in. J
.Bio
l.Che
m.
2005
. v28
0 p1
5202
Ban
ci, L
., et
al.
A h
int f
or t
he fu
nctio
n of
hum
an S
col
from
diff
eren
t st
ruct
ures
. Pro
c.N
atl.A
cad.
Sci
.Usa
200
6.
v103
p85
95
Ban
ci, L
., et
al.
A h
int f
or th
e fu
nctio
n of
hum
an S
col
from
diff
eren
t st
ruct
ures
. Pro
c.N
atl.A
cad.
Sci
.Usa
200
6.
v103
p85
95
Ban
ci, L
., et
al.
A h
int f
or th
e fu
nctio
n of
hum
an S
col
from
diff
eren
t st
ruct
ures
. Pro
c.N
atl.A
cad.
Sci
.Usa
200
6.
v103
pa5
95
2GT
6
2GQ
K
2GQ
L
2GG
T
1 FX
3
1 QY
N
2E50
1 L4l
1 JY
O
1 K3S
Sc
ol
(C-t
erm
inal
do
mai
n) -
Cu'
m
etal
ion
tran
spor
t N
MR
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
nla
nla
nla
2.4
2.5
2.35
2.3
2.2
1.9
1.9
Hom
o sa
pien
s B
anci
, L.
, et
al. A
hin
t for
the
func
tion
of h
uman
Sco
l fr
om d
iffer
ent s
truc
ture
s. P
roc.
Nat
l.Aca
d.S
ci.U
sa 2
006.
v1
03 p
8595
Ban
ci,
L., e
t al
. A
hin
t for
the
func
tion
of h
uman
Sco
l fr
om d
iffer
ent
stru
ctur
es.
Pro
c.N
atl.A
cad.
Sci
.Usa
200
6.
v103
p85
95
Ban
ci,
L., e
t al
. A
hin
t for
the
func
tion
of h
uman
Sco
l fr
om d
iffer
ent
stru
ctur
es.
Pro
c.N
atl.A
cad.
Sci
. Usa
200
6.
v103
p85
95
Ban
ci,
L.,
et a
l. S
col:
hin
ts fo
r th
e fu
nctio
n fr
om
stru
ctur
es.
To
be
pub
lishe
d.
Sc
ol
(C-t
erm
inal
do
mai
n) -
~i*
' m
etal
ion
tran
spor
t H
omo
sapi
ens
Sc
ol
(C-t
erm
inal
do
mai
n) -
~i
"
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort?
prot
ein
tran
spor
t
Hom
o sa
pien
s
Sco
l ho
mol
og -
~i*
' H
omo
sapi
ens
Sec
B
Hae
mop
hilu
s in
fluen
zae
Xu,
Z.,
Kna
fels
, J.D
., Y
oshi
no,
K.
Cry
stal
str
uctu
re o
f the
ba
cter
ial p
rote
in e
xpor
t ch
aper
one
secB
. N
at.S
truc
t.Bio
l. 20
00.
v7 p
1172
Sec
B
prot
ein
tran
spor
t E
sche
richi
a co
li D
ekke
r, C
., de
Kru
ijff,
B.,
Gro
s, P
. Cry
stal
str
uctu
re o
f S
ecB
from
Esc
heric
hia
coli.
J.S
truc
t.Bio
l. 20
03.
v144
p3
13
hist
one
chap
eron
e H
omo
sapi
ens
Mut
o, S
., et
al.
Rel
atio
nshi
p be
twee
n th
e st
ruct
ure
of
SE
TIT
AF
-Ibe
taIlN
HA
T a
nd it
s hi
ston
e ch
aper
one
activ
ity.
Pro
c.N
atl.A
cad.
Sci
.Usa
200
7. v
104
p428
5
Sfa
E
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
type
Ill
prot
ein
secr
etio
n
Esc
heric
hia
coli
Kni
ght,
S.D
., et
al.
Str
uctu
re o
f the
S p
ilus
perip
lasm
ic
chap
eron
e S
faE
at
2.2
A r
esol
utio
n. A
cta
Cry
stal
logr
., S
ect.
D 2
002.
v58
p10
16
Sic
P -
Spt
P c
ompl
ex
Sal
mon
ella
ty
phim
uriu
m
Sal
mon
ella
en
teric
a
Ste
bbin
s, C
.E.,
Gal
an, J
.E. M
aint
enan
ce o
f an
unfo
lded
po
lype
ptid
e by
a c
ogna
te c
hape
rone
in b
acte
rial t
ype
Ill
secr
etio
n. N
atur
e 20
01. v
414
p77
Luo,
Y.,
et a
l. S
truc
tura
l and
bio
chem
ical
cha
ract
eriz
atio
n of
the
type
Ill
secr
etio
n ch
aper
ones
Ces
T a
nd S
igE
. N
at.S
truc
t.Bio
l. 20
01. v
8 p1
031
Sig
E
type
Ill
prot
ein
secr
etio
n
1SG
2 S
kp
1U2M
S
kp
1 SH
S
1 M7K
1 RY
9
1 M5Y
1 JY
A
1 K6Z
1 N5B
smal
l he
at s
hock
pr
otei
ns
SO
DD
(B
AG
Dom
ain)
Sur
A
Syc
E
Syc
E
Syc
E
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
prev
entio
n of
pr
otei
n ag
greg
atio
n
apop
tosi
s
prot
ein
secr
etio
n
prot
ein
fold
ing
type
Ill
prot
ein
secr
etio
n
type
Ill
prot
ein
secr
etio
n
type
Ill
prot
ein
secr
etio
n
X-r
ay
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Esc
heric
hia
coli
Kor
ndor
fer,
I.P
., D
omm
el, M
.K.,
Ske
rra,
A.
Str
uctu
re o
f th
e pe
ripla
smic
cha
pero
ne S
kp s
ugge
sts
func
tiona
l si
mila
rity
with
cyt
osol
ic c
hape
rone
s de
spite
diff
erin
g ar
chite
ctur
e. N
at.S
truc
t.Mol
.Bio
l. 20
04.
vl 1
p10
15
Esc
heric
hia
coli
Wal
ton,
T.A
., S
ousa
, M.C
. Cry
stal
Str
uctu
re o
f S
kp, a
P
refo
ldin
-like
Cha
pero
ne th
at P
rote
cts
Sol
uble
and
M
embr
ane
Pro
tein
s fr
om A
ggre
gatio
n. M
oLC
ell 2
004.
v1
5 p3
67
Met
hano
cocc
us
Kim
, K
.K.,
Kim
, R
., K
im, S
.H.
Cry
stal
str
uctu
re o
f a s
mal
l ja
nnas
chii
heat
-sho
ck p
rote
in.
Nat
ure
1998
. v39
4 p5
95
Hom
o sa
pien
s
Shi
gella
flex
neri
Esc
h er
ichi
a co
li
Yer
sini
a ps
eudo
tube
rcul
os
is
Yer
sini
a p
est
is
Yer
sini
a en
tero
colit
ica
Bro
ckm
ann,
C.,
et a
l. T
he s
olut
ion
stru
ctur
e of
the
SO
DD
B
AG
dom
ain
reve
als
addi
tiona
l ele
ctro
stat
ic in
tera
ctio
ns
in th
e H
SP
70 c
ompl
exes
of
SO
DD
sub
fam
ily B
AG
do
mai
ns. F
EB
S L
ett.
2004
. v55
8 p1
01
van
Eer
de, A
,, et
al.
Str
uctu
re o
f Spa
1 5, a
type
Ill
secr
etio
n ch
aper
one
from
Shi
gella
flex
neri
with
bro
ad
spec
ifici
ty. E
MB
O R
ep. 2
004.
v5
p477
Bitt
o, E
., M
cKay
, D
.B. C
ryst
allo
grap
hic
Str
uctu
re o
f Sur
A,
a M
olec
ular
Cha
pero
ne th
at F
acili
tate
s F
oldi
ng o
f O
uter
M
embr
ane
Por
ins.
Str
uctu
re 2
002.
v10
p14
89
Birt
alan
, S.,
Gho
sh, P
. Str
uctu
re o
f the
Yer
sini
a ty
pe Ill
secr
etor
y sy
stem
cha
pero
ne S
ycE
. N
at.S
truc
t.Bio
l. 20
01.
v8 p
974
Evd
okim
ov, A
.G.,
et a
l. T
hree
-dim
ensi
onal
str
uctu
re o
f th
e ty
pe I
ll se
cret
ion
chap
eron
e S
ycE
from
Yer
sini
a pe
stis
. Act
a C
ryst
allo
gr.,
Sec
t.D 2
002.
v58
p39
8
Tra
me,
C.B
., M
cKay
, D.B
. Str
uctu
re o
f the
Yer
sini
a en
tero
colit
ica
Mol
ecul
ar-C
hape
rone
Pro
tein
Syc
E. A
cta
Cry
stal
logr
., S
ect.D
200
3. v
59 p
389
1 L2W
S
ycE
- Y
opE
ty
pe I
ll pr
otei
n (c
hape
rone
-bin
ding
se
cret
ion
dom
ain)
com
plex
X-r
ay
Yer
sini
a B
irtal
an,
S.C
., P
hilli
ps, R
.M.,
Gho
sh, P
. Thr
ee-
pseu
dotu
berc
ul
dim
ensi
onal
sec
retio
n si
gnal
s in
cha
pero
ne-e
ffect
or
osis
co
mpl
exes
of
bact
eria
l pat
hoge
ns.
Mol
.Cel
l 200
2. v
9 p9
71
1 TlW
S
ycH
- Y
scM
2 ty
pe I
ll pr
otei
n co
mp
lex
secr
etio
n X
-ray
Y
ersi
nia
pe
stis
P
han,
J.,
Tro
pea,
J.E
., W
augh
, D
.S.
Str
uctu
re o
f th
e C
09
2
Yer
sini
a pe
stis
type
Ill
secr
etio
n ch
aper
one
Syc
H in
co
mpl
ex w
ith a
sta
ble
frag
men
t of
Ysc
M2.
Act
a C
ryst
allo
gr.,
Sec
t.D n
la.
v60
p159
1
lXK
P
Syc
N-Y
scB
- Y
op
N
type
Ill
prot
ein
com
ple
x se
cret
ion
X-r
ay
Yer
sini
a pe
stis
S
chub
ot,
F.D
., et
al.
Thr
ee-d
imen
sion
al s
truc
ture
of
a m
acro
mol
ecul
ar a
ssem
bly
that
reg
ulat
es ty
pe I
ll se
cret
ion
in Y
ersi
nia
pest
is. J
.Mol
.Bio
l. 20
05. v
346
pll
47
Loch
er,
M.,
et a
l. C
ryst
al S
truc
ture
of t
he Y
ersi
nia
Ent
eroc
oliti
ca T
ype
Ill S
ecre
tion
Cha
pero
ne S
yct.
J.B
iol.C
hem
. 20
05.
v280
p31
149
But
tner
, C
.R.,
et a
l. C
ryst
al S
truc
ture
of Y
ersi
nia
Ent
eroc
oliti
ca T
ype
Ill S
ecre
tion
Cha
pero
ne S
yct.
Pro
tein
S
ci.
2005
. v14
p19
93
But
tner
, C
.R.,
et a
l. C
ryst
al S
truc
ture
of Y
ersi
nia
Ent
eroc
oliti
ca T
ype
Ill S
ecre
tion
Cha
pero
ne S
yct.
Pro
tein
S
ci.
2005
. v1
4 p1
993
But
tner
, C
.R.,
et a
l. C
ryst
al S
truc
ture
of Y
ersi
nia
Ent
eroc
oliti
ca T
ype
Ill S
ecre
tion
Cha
pero
ne S
yct.
Pro
tein
S
ci.
2005
. v1
4 p1
993
Ditz
el,
L.,
et a
l. C
ryst
al s
truc
ture
of
the
ther
mos
ome,
the
ar
chae
al c
hape
roni
n an
d ho
mol
og o
f CC
T. C
ell 1
998.
v9
3 p
12
5
Sho
mur
a, Y
., et
al.
Cry
stal
Str
uctu
res
of t
he G
roup
I1
Cha
pero
nin
from
The
rmoc
occu
s st
rain
KS
-1:
Ste
ric
Hin
dran
ce b
y th
e S
ubst
itute
d A
min
o A
cid,
and
Int
er-
subu
nit
Rea
rran
gem
ent
betw
een
Tw
o C
ryst
al F
orm
s.
J.M
ol.B
iol.
2004
. v3
35 p
1265
2B
H0
S
ycT
ty
pe I
ll pr
otei
n se
cret
ion
X-r
ay
Yer
sini
a en
tero
colit
ica
2BS
H
Syc
T
type
Ill
prot
ein
secr
etio
n X
-ray
Y
ersi
nia
ente
roco
litic
a
2BS
I S
ycT
ty
pe I
ll pr
otei
n se
cret
ion
Yer
sini
a en
tero
colit
ica
X-r
ay
2BS
J S
ycT
ty
pe I
ll pr
otei
n se
cret
ion
X-r
ay
Yer
sini
a en
tero
colit
ica
1A6D
T
herm
osom
e pr
otei
n fo
ldin
g T
herm
opla
sma
acid
ophi
lum
X
-ray
1Q2V
T
herm
osom
e (a
lpha
pr
otei
n fo
ldin
g su
buni
t, m
utan
t)
X-r
ay
The
rmoc
occu
s SP
.
1Q3R
T
herm
osom
e (a
lpha
pr
otei
n fo
ldin
g su
buni
t, m
utan
t)
1Q3S
T
herm
osom
e (a
lpha
pr
otei
n fo
ldin
g su
buni
t, m
utan
t) -
AD
P
1Q3Q
T
herm
osom
e (a
lpha
pr
otei
n fo
ldin
g su
buni
t, m
utan
t) -
AM
P-P
NP
IAS
S
The
rmos
ome
(api
cal
prot
ein
fold
ing
dom
ain)
, alp
ha s
ubun
it
IAS
X
The
rmos
ome
(api
cal
prot
ein
fold
ing
dom
ain)
, al
pha
subu
nit
1 EO
R
The
rmos
ome
(api
cal
prot
ein
fold
ing
dom
ain)
, be
ta s
ubun
it
1A6E
T
herm
osom
e-M
gG-
prot
ein
fold
ing
AD
P-A
LF3
com
plex
2GU
Z
Tim
14 -
Tim
16
co-c
hape
rone
co
mpl
ex
to
mito
chon
dria
l hs
p70
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
The
rmoc
occu
s SP
.
The
rmoc
occu
s SP
.
The
rmoc
occu
s SP
.
The
rmop
lasm
a ac
idop
hilu
m
The
rmop
lasm
a ac
idop
hilu
m
The
rmop
lasm
a ac
idop
hilu
m
The
rmop
lasm
a ac
idop
hilu
m
Sac
char
omyc
es
cere
visi
ae
Sho
mur
a, Y
., et
al.
Cry
stal
Str
uctu
res
of t
he G
roup
II
Cha
pero
nin
from
The
rmoc
occu
s st
rain
KS
-1: S
teric
H
indr
ance
by
the
Sub
stitu
ted
Am
ino
Aci
d, a
nd In
ter-
su
buni
t Rea
rran
gem
ent b
etw
een
Tw
o C
ryst
al F
orm
s.
J.M
ol.B
iol.
2004
. v3
35 p
1265
Sho
mur
a, Y
., et
al.
Cry
stal
Str
uctu
res
of t
he G
roup
II
Cha
pero
nin
from
The
rmoc
occu
s st
rain
KS
-1: S
teric
H
indr
ance
by
the
Sub
stitu
ted
Am
ino
Aci
d, a
nd I
nter
- su
buni
t Rea
rran
gem
ent b
etw
een
Tw
o C
ryst
al F
orm
s.
J.M
ol.B
iol.
2004
. v33
5 p1
265
Sho
mur
a, Y
., et
al.
Cry
stal
Str
uctu
res
of t
he G
roup
II
Cha
pero
nin
from
The
rmoc
occu
s st
rain
KS
-1: S
teric
H
indr
ance
by
the
Sub
stitu
ted
Am
ino
Aci
d, a
nd I
nter
- su
buni
t R
earr
ange
men
t bet
wee
n T
wo
Cry
stal
For
ms.
J.
Mol
.Bio
l. 20
04. v
335
p126
5
Klu
mpp
, M
., B
aum
eist
er, W
., E
ssen
, L.O
. S
truc
ture
of
the
subs
trat
e bi
ndin
g do
mai
n of
the
ther
mos
ome,
an
arch
aeal
gro
up I
I cha
pero
nin.
Cel
l 199
7. v
91 p
263
Klu
mpp
, M.,
Bau
mei
ster
, W.,
Ess
en, L
.O. S
truc
ture
of
the
subs
trat
e bi
ndin
g do
mai
n of
the
ther
mos
ome,
an
arch
aeal
gro
up I
I cha
pero
nin.
Cel
l 199
7. v
91 p
263
Bos
ch, G
., B
aum
eist
er, W
., E
ssen
, L.O
. Cry
stal
str
uctu
re
of th
e be
ta-a
pica
l dom
ain
of th
e th
erm
osom
e re
veal
s st
ruct
ural
pla
stic
ity in
the
prot
rusi
on re
gion
. J.M
ol.B
iol.
2000
. v3
01 p
1 9
Ditz
el, L
., et
al.
Cry
stal
str
uctu
re o
f the
ther
mos
ome,
the
ar
chae
al c
hape
roni
n an
d ho
mol
og o
f CC
T.
Cel
l 199
8.
v93
p125
Mok
ranj
ac, D
., et
al.
Str
uctu
re a
nd fu
nctio
n of
Tim
14 a
nd
Tim
l6,
the
J an
d J-
like
com
pone
nts
of t
he m
itoch
ondr
ial
prot
ein
impo
rt m
otor
. EM
BO
J.
2006
. v25
p46
75
2BS
K
Tim
9 - T
im10
com
plex
m
itoch
ondr
ia1
inte
rmem
bran
e
spac
e ch
aper
one
com
plex
INIC
T
orD
ch
aper
one
cofa
ctor
- de
pend
ent
prot
ein
fold
ing
1 H6
Q
Tra
nsla
tiona
lly
nla
co
ntro
lled
tum
or-
asso
ciat
ed p
rote
in
P2
3f~
p
(TC
TP
)
1 H7Y
T
rans
latio
nally
n
la
cont
rolle
d tu
mor
- as
soci
ated
pro
tein
p
23
f~~
(T
CT
P)
1W26
T
rigge
r F
acto
r pr
otei
n fo
ldin
g,
prot
ein
tran
spor
t
2N
SA
T
rigge
r F
acto
r pr
otei
n fo
ldin
g,
prot
ein
tran
spor
t
2NS
B
Trig
ger
Fac
tor
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
2NS
C
Trig
ger
Fac
tor
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
X-r
ay
X-r
ay
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Hom
o sa
pien
s W
ebb,
C.T
., et
al.
Cry
stal
Str
uctu
re o
f the
Mito
chon
dria
1 C
hape
rone
Tim
910
Rev
eals
a S
ix-B
lade
d A
lpha
- P
rope
ller.
Mol
.Cel
l 200
6. v
21 p
123
She
wan
ella
m
assi
lia
Tra
nier
, S
., et
al.
A N
ovel
Pro
tein
Fol
d an
d E
xtre
me
Dom
ain
Sw
appi
ng in
the
Dim
eric
Tor
D C
hape
rone
from
S
hew
anel
la m
assi
lia. S
truc
ture
200
3. v
l I p
165
Sch
izos
acch
aro
Tha
w,
P.,
et a
l. S
truc
ture
of T
CT
P r
evea
ls u
nexp
ecte
d m
yce
s p
om
be
re
latio
nshi
p w
ith g
uani
ne n
ucle
otid
e-fr
ee c
hape
rone
s.
Nat
.Str
uct.B
iol.
2001
. v8
p70
1
Sch
izos
acch
aro
Tha
w,
P.,
et a
l. S
truc
ture
of T
CT
P r
evea
ls u
nexp
ecte
d m
yces
po
mb
e
rela
tions
hip
with
gua
nine
nuc
leot
ide-
free
cha
pero
nes.
N
at.S
truc
t.Bio
l. 20
01.
v8 p
701
Esc
heric
hia
coli
Fer
bitz
, L.,
et a
l. T
rigge
r F
acto
r in
Com
plex
with
the
R
ibos
ome
For
ms
a M
olec
ular
Cra
dle
for
Nas
cent
P
rote
ins.
Nat
ure
2004
. v4
31 p
590
The
rmot
oga
Mar
tinez
-Hac
kert
, E.,
Hen
dric
kson
, W.A
. S
truc
ture
s of
m
ariti
ma
and
inte
ract
ions
bet
wee
n do
mai
ns o
f tr
igge
r fa
ctor
from
T
hem
otog
a m
ariti
ma.
Act
a C
ryst
allo
gr.,S
ect.D
200
7.
vD63
p53
6
The
rmot
oga
Mar
tinez
-Hac
kert
, E.,
Hen
dric
kson
, W.A
. Str
uctu
res
of
mar
itim
a an
d in
tera
ctio
ns b
etw
een
dom
ains
of
trig
ger
fact
or fr
om
The
mot
oga
mar
itim
a. A
cta
Cry
stal
logr
.,Sec
t.D 2
007.
vD
63 p
536
The
rmot
oga
Mar
tinez
-Hac
kert
, E.,
Hen
dric
kson
, W.A
. S
truc
ture
s of
m
ariti
ma
and
inte
ract
ions
bet
wee
n do
mai
ns o
f tr
igge
r fa
ctor
from
T
hem
otog
a m
ariti
ma.
Act
a C
ryst
allo
gr.,S
ect.D
200
7.
vD63
p53
6
2AA
R
Trig
ger
Fac
tor
- rib
osom
e fr
agm
ent
com
plex
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
X-r
ay
Dei
noco
ccus
R
adio
dura
ns
Bar
am, D
., et
al.
Str
uctu
re o
f trig
ger
fact
or b
indi
ng
dom
ain
in b
iolo
gica
lly h
omol
ogou
s co
mpl
ex w
ith
euba
cter
ial
ribos
ome
reve
als
its c
hape
rone
act
ion.
P
roc.
Nat
l.Aca
d.S
ci.U
sa 2
005.
v10
2 p1
2017
1 HX
V
Trig
ger
Fac
tor
(PP
IAse
do
mai
n)
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
NM
R
Myc
opla
sma
geni
taliu
m
Vog
ther
r, M
., et
al.
NM
R s
olut
ion
stru
ctur
e an
d dy
nam
ics
of th
e pe
ptid
yl-p
roly
l cis
-tra
ns is
orne
rase
dom
ain
of th
e tr
igge
r fa
ctor
fro
m M
ycop
lasm
a ge
nita
lium
com
pare
d to
F
K50
6-bi
ndin
g pr
otei
n. J
.Mol
.Bio
l. 20
02. v
318
p109
7
10
MS
T
rigge
r F
acto
r (R
ibos
ome
bind
ing
dom
ain)
1 P9Y
T
rigge
r F
acto
r (r
ibos
ome-
bind
ing
dom
ain,
mut
ant)
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
NM
R
NM
R
2.3
2.15
2.5
3.5
2.5
1 .8
n/a
nla
Esc
heric
hia
coli
Kris
tens
en,
O.,
Gaj
hede
, M
. Cha
pero
ne B
indi
ng a
t the
R
ibos
omal
Exi
t Tun
nel.
Str
uctu
re 2
003.
vl I p
1547
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
Esc
heric
hia
coli
Kris
tens
en, O
., G
ajhe
de,
M. C
hape
rone
Bin
ding
at t
he
Rib
osom
al E
xit T
unne
l. S
truc
ture
200
3. v
l I p
1547
1 TI 1
T
rigge
r F
acto
r (t
runc
ated
) pr
otei
n fo
ldin
g,
prot
ein
tran
spor
t
Vib
rio c
hole
rae
Ludl
am, A
.V.,
Moo
re, B
.A.,
Xu,
Z. T
he c
ryst
al s
truc
ture
of
ribos
omal
cha
pero
ne tr
igge
r fa
ctor
from
Vib
rio c
hole
rae.
P
roc.
Nat
l.Aca
d.S
ci.U
SA
200
4. v
l01
p13
436
1 W2
B
Trig
ger
Fac
tors
(N
- te
rmin
al d
omai
n) -
rib
osom
e co
mpl
ex
2BO
L T
SP
36
prot
ein
fold
ing,
pr
otei
n tr
ansp
ort
Hal
oarc
ula
mar
ism
ortu
i F
erbi
tz, L
., et
al.
Trig
ger
Fac
tor
in C
ompl
ex w
ith t
he
Rib
osom
e F
orm
s a
Mol
ecul
ar C
radl
e fo
r N
asce
nt
Pro
tein
s. N
atur
e 20
04. v
431
p590
smal
l hea
t sh
ock
prot
ein
Tae
nia
sagi
nata
S
tam
ler,
R.,
et a
l. W
rapp
ing
the
Alp
ha-C
ryst
allin
Dom
ain
Fol
d in
a C
hape
rone
Ass
embl
y. J
.Mol
.Bio
l. 20
05. v
353
~6
8
1 H7
C
tubu
lin c
hape
rone
co
fact
or A
(C
oA)
tubu
lin fo
ldin
g H
omo
sapi
ens
Gua
sch,
A.,
et a
l. T
hree
-dim
ensi
onal
str
uctu
re o
f hu
man
tu
bulin
cha
pero
ne c
ofac
tor
A.
J.M
ol.B
iol.
2002
. v
3l8
p1
139
1 WH
G
tubu
lin s
peci
fic
chap
eron
e B
(C
AP
-Gly
do
mai
n)
Mus
mus
culu
s S
aito
, K
., et
al.
Sol
utio
n st
ruct
ure
of th
e C
AP
-Gly
dom
ain
in m
ouse
tubu
lin s
peci
fic c
hape
rone
€3.
To
be p
ublis
hed
. V
P
1 WJN
tu
bulin
spe
cific
ch
aper
one
E (
C-
term
inal
dom
ain)
Mus
mus
culu
s S
ato,
M.,
et a
l. S
olut
ion
stru
ctur
e of
the
C-t
erm
inal
ub
iqui
tin-li
ke d
omai
n of
mou
se tu
bulin
-spe
cific
ch
aper
one
e. T
o be
pub
lishe
d.
1TO
Y
Tub
ulin
-bm
ding
C
ofac
tor
B (
Ubi
quiti
n-
like
dom
ain)
1V6E
T
ubul
in-s
peci
fic
Cha
pero
ne B
(N
- te
rmin
al d
omai
n)
1 JM
V
Uni
vers
al S
tres
s P
rote
in
1 GM
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Ure
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cu2
'
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R
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E -
zn2'
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E -
zn2'
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whe
at s
HS
P 1
6.9
10
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W
ind
2CO
E
Win
d
2CO
F W
ind
(m
utan
t)
tubu
lin fo
ldin
g
tubu
lin f
oldi
ng
resp
onse
to
stre
ss
met
al io
n bi
ndin
g
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
prot
ein
secr
etio
n, P
DI-
re
late
d
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
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ay
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ay
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ay
X-r
ay
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ay
n/a
Cae
norh
abdi
tis
eleg
ans
n/a
M
us m
uscu
lus
1.85
H
aem
ophi
lus
influ
enza
e
1.5
Kle
bsie
lla
aero
gene
s
1.7
Bac
illus
p
ast
eu
rii
1.85
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acill
us
past
euri
i
2.7
Trit
icum
ae
stiv
um
1 .9
D
roso
phila
m
elan
ogas
ter
2.35
D
roso
phila
m
elan
ogas
ter
2.28
D
roso
phila
m
elan
ogas
ter
Lytle
, B
.L.,
et a
l. S
olut
ion
Str
uctu
re o
f a
Ubi
quiti
n-lik
e D
omai
n fr
om T
ubul
in-b
indi
ng C
ofac
tor
B. J
.Bio
l.Che
m.
2004
. v2
79 p
4678
7
Zha
o, C
., et
al.
Sol
utio
n S
truc
ture
of
a N
-ter
min
al
Ubi
quiti
n-lik
e D
omai
n in
Mou
se T
ubul
in-s
peci
fic
Cha
pero
ne B
. To
be
pub
lishe
d.
Sou
sa,
M.C
., M
cKay
, D.B
. Str
uctu
re o
f the
uni
vers
al
stre
ss p
rote
in o
f H
aem
ophi
lus
influ
enza
e. S
truc
ture
20
01.
v9 p
ll3
5
Son
g, H
.K.,
et a
l. C
ryst
al s
truc
ture
of
Kle
bsie
lla
aero
gene
s U
reE
, a n
icke
l-bin
ding
met
allo
chap
eron
e fo
r ur
ease
act
ivat
ion.
J.B
iol.C
hem
. 20
01.
v276
p49
359
Rem
aut,
H.,
et a
l. S
truc
tura
l bas
is fo
r N
i(2+
) tr
ansp
ort
and
asse
mbl
y of
the
ure
ase
activ
e si
te b
y th
e m
etal
loch
aper
one
Ure
E fr
om B
acill
us p
aste
urii.
J.
Bio
l.Che
m.
2001
. v2
76 p
4936
5
Rem
aut,
H.,
et a
l. S
truc
tura
l bas
is fo
r N
i(2+
) tr
ansp
ort
and
asse
mbl
y of
the
ure
ase
activ
e si
te b
y th
e
met
allo
chap
eron
e U
reE
from
Bac
illus
pas
teur
ii.
J.B
iol.C
hem
. 200
1. v
276
p493
65
van
Mon
tfort
, R
.L.,
et a
l. C
ryst
al s
truc
ture
and
ass
embl
y of
a e
ukar
yotic
sm
all h
eat s
hock
pro
tein
. Nat
.Str
uct.B
iol.
2001
. v8
p10
25
Ma,
Q.,
et a
l. C
ryst
al s
truc
ture
and
func
tiona
l ana
lysi
s of
D
roso
phila
Win
d, a
pro
tein
-dis
ulfid
e is
omer
ase-
rela
ted
prot
ein.
J.B
iol.C
hem
. 200
3. v
278
p446
00
Sev
vana
, M
., et
al.
Str
uctu
ral E
luci
datio
n of
the
Pdi
- R
elat
ed C
hape
rone
Win
d w
ith th
e H
elp
of M
utan
ts. A
cta
Cry
stal
logr
.,Sec
t.D 2
006.
v62
p58
9
Sev
vana
, M
., et
al.
Str
uctu
ral
Elu
cida
tion
of th
e P
di-
Rel
ated
Cha
pero
ne W
ind
with
the
Hel
p of
Mut
ants
. Act
a C
ryst
allo
gr.,S
ect.D
200
6. v
62 p
589
Win
d (m
utan
t)
Win
d (m
utan
t)
yeas
t co
pper
ch
aper
one
for
supe
roxi
de d
ism
utat
e (d
omai
n 2
) - c
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al io
n tr
ansp
ort
type
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prot
ein
secr
etio
n
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ay
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ay
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ay
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ay
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ay
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omyc
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visi
ae
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mon
ella
ty
phim
uriu
m
Esc
heri
chia
co
li
Yer
sini
a pe
stis
Sev
vana
, M.,
et a
l. S
truc
tura
l Elu
cida
tion
of t
he P
di-
Rel
ated
Cha
pero
ne W
ind
with
the
Hel
p of
Mut
ants
. Act
a C
ryst
allo
gr.,S
ect.D
200
6. v
62 p
589
Sev
vana
, M
., e
t al
. S
truc
tura
l Elu
cida
tion
of t
he P
di-
Rel
ated
Cha
pero
ne W
ind
with
the
Hel
p of
Mut
ants
. Act
a C
ryst
allo
gr.,S
ect.D
200
6. v
62 p
589
Ach
ila,
D.,
et a
l. S
truc
ture
of
hum
an W
ilson
pro
tein
do
mai
ns 5
and
6 a
nd th
eir
inte
rpla
y w
ith d
omai
n 4
and
the
copp
er c
hape
rone
HA
HI
in c
oppe
r up
take
. P
roc.
Nat
l.Aca
d.S
ci.U
sa 2
006.
v10
3 p5
729
Hal
l, L.
T.,
et a
l. X
-ray
cry
stal
logr
aphi
c an
d an
alyt
ical
ul
trac
entr
ifuga
tion
anal
yses
of t
runc
ated
and
full-
leng
th
yeas
t co
pper
cha
pero
nes
for
SO
D (
LYS
7): a
dim
er-d
imer
m
odel
of
LYS
7-S
OD
ass
ocia
tion
and
copp
er d
eliv
ery.
B
ioch
emis
try
2000
. v3
9 p3
611
Nic
hols
, C.E
., et
al.
Str
uctu
ral C
hara
cter
izat
ion
of
Sal
mon
ella
typh
imur
ium
Yea
Z,
an
M22
0-
Sia
logl
ycop
rote
in E
ndop
eptid
ase
Hom
olog
. P
rote
ins
2006
. v6
4 p
l I I
Nic
hols
, C
.E.,
et a
l. S
truc
tura
l Cha
ract
eriz
atio
n of
S
alm
onel
la ty
phim
uriu
m Y
eaZ
, an
M22
0-
S
ialo
glyc
opro
tein
End
opep
tidas
e H
omol
og.
Pro
tein
s 20
06.
v64
pi I I
Zha
o, Y
., et
al.
The
cry
stal
str
uctu
re o
f E
sche
richi
a co
li he
at s
hock
pro
tein
Yed
U r
evea
ls t
hree
pot
entia
l cat
alyt
ic
activ
e si
tes.
Pro
tein
Sci
. 200
3. v
12 p
2303
Pha
n, J
., A
ustin
, B
.P.,
Wau
gh,
D.S
. C
ryst
al s
truc
ture
of
the
Yer
sini
a ty
pe I
ll se
cret
ion
prot
ein
Ysc
E.
Pro
tein
Sci
. 20
05. v
14 p
2759
REFERENCE LIST
Akeda Y & Galan JE (2005) Chaperone release and unfolding of substrates in type I I I secretion. Nature 437: 91 1-91 5
Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW, Prodromou C, & Pearl LH (2006) Crystal structure of an Hsp90-nucleotide-p23lSbal closed chaperone complex. Nature 440: 101 3-1 01 7
Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181 : 223-230
Arcus V (2002) OB-fold domains: a snapshot of the evolution of sequence, structure and function. Curr Opin Strucf Biol12: 794-801
Baars L, Ytterberg AJ, Drew D, Wagner S, Thilo C, van Wijk KJ, & de Gier JW (2006) Defining the role of the Escherichia coli chaperone SecB using comparative proteomics. J Biol Chem 281 : 10024-1 0034
Barnett ME, Nagy M, Kedzierska S, & Zolkiewski M (2005) The amino-terminal domain of ClpB supports binding to strongly aggregated proteins. J Biol Chem 280: 34940-34945
Barnhart MM, Pinkner JS, Soto GE, Sauer FG, Langermann S, Waksman G, Frieden C, & Hultgren SJ (2000) PapD-like chaperones provide the missing information for folding of pilin proteins. Proc Nafl Acad Sci U S A 97: 7709-7714
Bartolucci C, Lamba D, Grazulis S, Manakova E, & Heumann H (2005) Crystal structure of wild-type chaperonin GroEL. J Mol Biol354: 940-951
Beck K, Wu LF, Brunner J, & Muller M (2000) Discrimination between SRP- and SecNSecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor. EM60 J 19: 134-143
Behrens S, Maier R, de Cock H, Schmid FX, & Gross CA (2001) The SurA periplasmic PPlase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J 20: 285-294
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, & Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28: 235-242
Bernstein HD, Poritz MA, Strub K, Hoben PJ, Brenner S, &Walter P (1989) Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature 340: 482-486
Binkowski TA, Naghibzadeh S, & Liang J (2003) CASTp: Computed Atlas of Surface Topography of proteins. Nucleic Acids Res 31 : 3352-3355
Birtalan SC, Phillips RM, & Ghosh P (2002) Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell 9: 971- 980
Bitto E & McKay DB (2002) Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure 10: 1489-1 498
Bosl B, Grimminger V, & Walter S (2006) The molecular chaperone Hspl04--a molecular machine for protein disaggregation. J Sfruct Biol156: 139-148
Braig K, Adams PD, & Brunger AT (1 995) Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution. Nat Struct Biol2: 1083-1 094
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges MI Pannu NS, Read RJ, Rice LM, Simonson TI & Warren GL (1 998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: 905-92 1
Buchner J & Walter S (2005) Analysis of Chaperone Function in Vitro. In Protein Folding Handbook, Buchner J & Kiefhaber T (eds) pp 162-1 96. WlLEY - VCH Verlag GmbH & Co. KGaA: Weinheim
Bulieris PV, Behrens S, Holst 0 , & Kleinschmidt JH (2003) Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J Biol Chem 278: 9092-9099
Burston SG & Walter S (2005) The E.coli GroE Chaperone. In Protein Folding Handbook, Buchner J & Kiefhaber T (eds) pp 699-724. WILEY-VCH Verlag GmbH & Co.: KGaA, Weinheim
Capitani GI Eidam 0 , Glockshuber R, & Grutter MG (2006) Structural and functional insights into the assembly of type 1 pili from Escherichia cob. Microbes Infect 8 : 2284-2290
Chen L & Sigler PB (1999) The crystal structure of a GroELIpeptide complex: plasticity as a basis for substrate diversity. Cell 99: 757-768
Chen R & Henning U (1996) A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins. Mol Microbial 19: 1287-1 294
Choudhury D, Thompson A, Stojanoff V, Langermann S, Pinkner J, Hultgren SJ, & Knight SD (1999) X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli. Science 285: 1061 -1 066
Crane JM, Suo Y, Lilly A , , Mao C, Hubbell WL, & Randall LL (2006) Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling. J Mol Biol363: 63-74
Crepin T, Schmitt El Blanquet S, & Mechulam Y (2002) Structure and function of the C-terminal domain of methionyl-tRNA synthetase. Biochemistry 41: 13003-1 301 1
de Keyzer J, van der Does C, & Driessen AJ (2003) The bacterial translocase: a dynamic protein channel complex. Cell Mol Life Sci 60: 2034-2052
Dekker C, de Kruijff B, & Gros P (2003) Crystal structure of SecB from Escherichia coli. J Struct Biol l44: 31 3-31 9
DeLano WL (2002) The PyMOL Molecular Graphics System.
Deuerling El Patzelt HI Vorderwulbecke S, Rauch T, Kramer GI Schaffitzel E, Mogk A, Schulze-Specking A, Langen HI & Bukau B (2003) Trigger Factor and DnaK possess overlapping substrate pools and binding specificities. Mol Microbiol 47: 1 31 7-1 328
Deuerling El Schulze-Specking A, Tomoyasu TI Mogk A, & Bukau B (1999) Trigger factor and DnaK cooperate in folding of newly synthesized proteins. Nature 400: 693-696
Ditzel L, Lowe J, Stock Dl Stetter KO, Huber HI Huber R, & Steinbacher S (1998) Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell 93: 125-1 38
Dougan DA, Mogk A, Zeth K, Turgay K, & Bukau B (2002) AAA+ proteins and substrate recognition, it all depends on their partner in crime. FEBS Lett 529: 6-10
Driessen AJ, Manting EH, & van der Does C (2001) The structural basis of protein targeting and translocation in bacteria. Nat Struct Biol8: 492-498
Egea PF, Stroud RM, &Walter P (2005) Targeting proteins to membranes: structure of the signal recognition particle. Curr Opin Struct Biol15: 213- 220
Ellis J (1987) Proteins as molecular chaperones. Nature 328: 378-379
Ellis RJ & Minton AP (2006) Protein aggregation in crowded environments. Biol Chem 387: 485-497
Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2 126-2 132
Fekkes P & Driessen AJ (1999) Protein targeting to the bacterial cytoplasmic membrane. Microbiol Mol Biol Rev 63: 161 -1 73
Feldman DE, Spiess C, Howard DE, & Frydman J (2003) Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol Cell 12: 1213-1224
Feldman MF & Cornelis GR (2003) The multitalented type Ill chaperones: all you can do with 15 kDa. FEMS Microbial Left 21 9: 151 -1 58
Fenton WA, Kashi Y, Furtak K, & Horwich AL (1994) Residues in chaperonin GroEL required for polypeptide binding and release. Nature 371: 614-619
Ferbitz L, Maier T, Patzelt H, Bukau B, Deuerling E, & Ban N (2004) Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431 : 590-596
Flaherty KM, DeLuca-Flaherty C, & McKay DB (1 990) Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346: 623-628
Galan JE & Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type Ill secretion machines. Nature 444: 567-573
Gibrat JF, Madej T, & Bryant SH (1996) Surprising similarities in structure comparison. Curr Opin Struct Biol6: 377-385
Glaser F, Pupko T, Paz I, Bell RE, Bechor-Shental D, Martz E, & Ben-Tal N (2003) ConSurf: identification of functional regions in proteins by surface- mapping of phylogenetic information. Bioinformatics 19: 163-1 64
Gouet P, Robert X, & Courcelle E (2003) ESPriptlENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31 : 3320-3323
Guo F, Maurizi MR, Esser L, & Xia D (2002) Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J Biol Chem 277: 46743- 46752
Ha SC, Lee TH, Cha SS, & Kim KK (2004) Functional identification of the SecB homologue in Methanococcus jannaschii and direct interaction of SecB with trigger factor. Biochem Biophys Res Commun 31 5: 1039-1 044
Halic M & Beckmann R (2005) The signal recognition particle and its interactions during protein targeting. Curr Opin Struct Biol15: 1 16-125
Halic M, Blau M, Becker T, Mielke T, Pool MR, Wild K, Sinning I, & Beckmann R (2006) Following the signal sequence from ribosomal tunnel exit to signal recognition particle. Nature 444: 507-51 1
Harms N, Koningstein G, Dontje W, Muller M, Oudega 0, Luirink J, & de Cock H (2001) The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane. J Biol Chem 276: 18804-1 881 1
Hartl FU (1 996) Molecular chaperones in cellular protein folding. Nature 381: 57 1-579
Hartl FU & Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 1852-1858
Hennecke G, Nolte J, Volkmer-Engert R, Schneider-Mergener J, & Behrens S (2005) The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition. J Biol Chem 280: 23540-23548
Hennessy F, Nicoll WS, Zimmermann R, Cheetham ME, & Blatch GL (2005) Not all J domains are created equal: implications for the specificity of Hsp40- Hsp70 interactions. Protein Sci 14: 1697-1 709
Hernandez MP, Sullivan WP, & Toft DO (2002) The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex. J Biol Chem 277: 38294-38304
Hesterkamp T, Hauser S, Lutcke H, & Bukau B (1996) Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. Proc Natl Acad Sci U S A 93: 4437-4441
Hinnerwisch J, Fenton WA, Furtak KJ, Farr GW, & Horwich AL (2005) Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121 : 1029-1 041
Hoffmann A, Merz F, Rutkowska A, Zachmann-Brand B, Deuerling E, & Bukau B (2006) Trigger factor forms a protective shield for nascent polypeptides at the ribosome. J Biol Chem 281 : 6539-6545
Holmgren A & Branden CI (1989) Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. Nature 342: 248-251
Hooft RW, Sander C, & Vriend G (1996) Positioning hydrogen atoms by optimizing hydrogen-bond networks in protein structures. Proteins 26: 363-376
Hunt JF, Weaver AJ, Landry SJ, Gierasch L, & Deisenhofer J (1996) The crystal structure of the GroES co-chaperonin at 2.8 A resolution. Nature 379: 37- 45
Hynes GM & Willison KR (2000) Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin. J Biol Chem 275: 1 8985-1 8994
Hyyrylainen HL, Sarvas M, & Kontinen VP (2005) Transcriptome analysis of the secretion stress response of Bacillus subtilis. Appl Microbial Biotechnol 67: 389-396
Jiang J, Prasad K, Lafer EM, & Sousa R (2005) Structural basis of interdomain communication in the Hsc70 chaperone. Mol Cell 20: 51 3-524
Jones S & Thornton JM (1995) Protein-protein interactions: a review of protein dimer structures. Prog Biophys Mol 5iol63: 31-65
Kabsch W & Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22: 2577-2637
Kandror 0, Sherman M, Rhode M, & Goldberg AL (1995) Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins. EMBO J 14: 6021-6027
Kawaguchi S, Muller J, Linde D, Kuramitsu S, Shibata T, lnoue Y, Vassylyev DG, & Yokoyama S (2001) The crystal structure of the ttCsaA protein: an export-related chaperone from Thermus thermophilus. EMBO J 20: 562- 569
Keenan RJ, Freymann DM, Walter P, & Stroud RM (1998) Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94: 181-191
Keiler KC, Waller PR, & Sauer RT (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271 : 990-993
Kerner MJ, Naylor DJ, lshihama Y, Maier T, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-Hartl M, Mann M, & Hartl FU (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122: 209-220
Knoblauch NT, Rudiger S, Schonfeld HJ, Driessen AJ, Schneider-Mergener J, & Bukau B (1 999) Substrate specificity of the SecB chaperone. J Biol Chem 274: 3421 9-34225
Korndorfer IP, Dommel MK, & Skerra A (2004) Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture. Nat Struct Mol Biol 11 : 101 5-1 020
Kramer G, Patzelt H, Rauch T, Kurz TA, Vorderwulbecke S, Bukau B, & Deuerling E (2004) Trigger factor peptidyl-prolyl cisltrans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli. J 5iol Chem 279: 141 65-1 41 70
Kramer G, Rauch T, Rist W, Vorderwulbecke S, Patzelt H, Schulze-Specking A, Ban N, Deuerling E, & Bukau B (2002) L23 protein functions as a chaperone docking site on the ribosome. Nature 419: 171-174
Kuehn MJ, Normark S, & Hultgren SJ (1991) Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits. Proc Natl Acad Sci U S A 88: 10586-1 0590
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V et al (1 997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249-256
Laufen T, Mayer MP, Beisel C, Klostermeier D, Mogk A, Reinstein J, & Bukau B (1999) Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A 96: 5452-5457
Lee PA, Tullman-Ercek Dl & Georgiou G (2006) The bacterial twin-arginine translocation pathway. Annu Rev Microbiol 60: 373-395
Lee S, Sowa ME, Watanabe YH, Sigler PB, Chiu W, Yoshida M, & Tsai FT (2003) The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 11 5: 229-240
Leroux MR (2001) Protein folding and molecular chaperones in archaea. Adv Appl Microbiol 50: 2 1 9-277
Letzelter M, Sorg I, Mota LJ, Meyer S, Stalder J, Feldman M, Kuhn M, Callebaut I, & Cornelis GR (2006) The discovery of SycO highlights a new function for type Ill secretion effector chaperones. EM60 J 25: 3223-3233
Li J, Qian X, & Sha B (2003) The crystal structure of the yeast Hsp40 Ydjl complexed with its peptide substrate. Structure 1 1 : 1475-1483
Lin Z & Rye HS (2006) GroEL-mediated protein folding: making the impossible, possible. Crit Rev Biochem Mol Biol41: 21 1-239
Linde D, Volkmer-Engert R, Schreiber S, & Muller JP (2003) Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY. FEMS Microbiol Lett 226: 93-100
Llorca 0 , McCormack EA, Hynes G, Grantham J, Cordell J, Carrascosa JL, Willison KR, Fernandez JJ, & Valpuesta JM (1999) Eukaryotic type II chaperonin CCT interacts with actin through specific subunits. Nature 402: 693-696
Ludlam AV, Moore BA, & Xu Z (2004) The crystal structure of ribosomal chaperone trigger factor from Vibrio cholerae. Proc Natl Acad Sci U S A 101 : 13436-1 3441
Luirink J & Sinning 1 (2004) SRP-mediated protein targeting: structure and function revisited. Biochim Biophys Acta 1694: 17-35
Lund 0, Nielsen M, Lundegaard C, & Worning P (2002) CPHmodels 2.0: X3M a Computer Program to Extract 3D Models.
Luo Y, Bertero MG, Frey EA, Pfuetzner RA, Wenk MR, Creagh L, Marcus SL, Lim D, Sicheri F, Kay C, Haynes C, Finlay BB, & Strynadka NC (2001) Structural and biochemical characterization of the type Ill secretion chaperones CesT and SigE. Naf Sfrucf Biol8: 1031-1036
Maier T, Ferbitz L, Deuerling E, & Ban N (2005) A cradle for new proteins: trigger factor at the ribosome. Curr Opin Sfrucf Biol15: 204-212
Maiti R, Van Domselaar GH, Zhang H, & Wishart DS (2004) Superpose: a simple server for sophisticated structural superposition. Nucleic Acids Res 32: W 590-4
Martin-Benito J, Boskovic J, Gomez-Puertas P, Carrascosa JL, Simons CT, Lewis SA, Bartolini F, Cowan NJ, & Valpuesta JM (2002) Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. EMBO J 21: 6377-6386
Maurizi MR & Xia D (2004) Protein binding and disruption by ClplHspI 00 chaperones. Sfrucfure 12: 175-1 83
Mayer MP & Bukau B (2005a) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62: 670-684
Mayer MP & Bukau B (2005b) Regulation of Hsp70 Chaperones by Co- chaperones. In Profein Folding Handbook, Buchner J & Kiefhaber T (eds) pp 516-562. WILEY-VCH Verlag GmbH & Co.: KGaA, Weinheim
Mayer MP & Bukau B (1999) Molecular chaperones: the busy life of Hsp9O. Curr Biol9: R322-5
Mayer MP & Bukau B (1998) Hsp7O chaperone systems: diversity of cellular functions and mechanism of action. Biol Chem 379: 261-268
McCoy AJ, Grosse-Kunstleve RW, Storoni LC, & Read RJ (2005) Likelihood- enhanced fast translation functions. Acfa Crysfallogr D Biol Crysfallogr 61: 458-464
Merz F, Hoffmann A, Rutkowska A, Zachmann-Brand B, Bukau 8, & Deuerling E (2006) The C-terminal domain of Escherichia coli trigger factor represents the central module of its chaperone activity. J Biol Chem 281: 31963- 31971
Meyer AS, Gillespie JR, Walther D, Millet IS, Doniach S, & Frydman J (2003a) Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis. Cell 113: 369-381
Meyer P, Prodromou C, Hu 6, Vaughan C, Roe SM, Panaretou 6 , Piper PW, & Pearl LH (2003b) Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol Cell 11 : 647-658
Mitra K, Frank J, & Driessen A (2006) Co- and post-translational translocation through the protein-conducting channel: analogous mechanisms at work? Nat Struct Mol Biol13: 957-964
Morris AL, MacArthur MW, Hutchinson EG, & Thornton JM (1992) Stereochemical quality of protein structure coordinates. Proteins 12: 345- 364
Muller J, Walter F, van Dijl JM, & Behnke D (1992) Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis. Mol Gen Genet 235: 89-96
Muller JP, Bron S, Venema G, & van Dijl JM (2000a) Chaperone-like activities of the CsaA protein of Bacillus subtilis. Microbiology 146 ( Pt 1): 77-88
Muller JP, Ozegowski J, Vettermann S, Swaving J, Van Wely KH, & Driessen AJ (2000b) Interaction of Bacillus subtilis CsaA with SecA and precursor proteins. Biochem J 348 Pt 2: 367-373
Murshudov GN, Vagin AA, & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Ctystallogr D Biol Crystallogr 53: 240-255
Murzin AG (1993) OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12: 861 -867
O'Brien MC, Flaherty KM, & McKay DB (1996) Lysine 71 of the chaperone protein Hsc70 Is essential for ATP hydrolysis. J Biol Chem 271 : 15874- 15878
Okochi M, Nomura 1, Zako 1 , Arakawa 1 , lizuka R, Ueda H, Funatsu T, Leroux M, & Yohda M (2004) Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin. J Biol Chem 279: 31 788-31 795
Painter J & Merritt EA (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr D Biol Ctystallogr 62: 439-450
Palmer 1 , Sargent F, & Berks BC (2005) Export of complex cofactor-containing proteins by the bacterial Tat pathway. Trends Microbial 13: 175-1 80
Parsot C, Hamiaux C, & Page AL (2003) The various and varying roles of specific chaperones in type Ill secretion systems. Curr Opin Microbiol 6: 7- 14
Patzelt H, Rudiger S, Brehmer D, Kramer G, Vorderwulbecke S, Schaffitzel E, Waitz A, Hesterkamp T, Dong L, Schneider-Mergener J, Bukau B, & Deuerling E (2001) Binding specificity of Escherichia coli trigger factor. Proc Natl Acad Sci U S A 98: 14244- 14249
Pearl LH & Prodromou C (2006) Structure and mechanism of the Hsp9O molecular chaperone machinery. Annu Rev Biochem 75: 271-294
Pflugrath JW (1999) The finer things in X-ray diffraction data collection. Acta Crystallogr D Biol Crysfallogr 55: 1 7 1 8- 1 725
Piatek R, Zalewska B, Bury K, & Kur J (2005) The chaperone-usher pathway of bacterial adhesin biogenesis -- from molecular mechanism to strategies of anti-bacterial prevention and modern vaccine design. Acfa Biochim Pol 52: 639-646
Picard D (2002) Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci 59: 1640-1 648
Pohlschroder M, Hartmann El Hand NJ, Dilks K, & Haddad A (2005) Diversity and evolution of protein translocation. Annu Rev Microbiol59: 91-1 11
Prescott LM, Harley JP, & Klein DA (2002) Microbiology. McGraw-Hill Higher Education: New York, NY, USA
Qiu XB, Shao YM, Miao S, & Wang L (2006) The diversity of the DnaJIHsp40 family, the crucial partners for Hsp70 chaperones. Cell Mol Life Sci 63: 2560-2570
Ramu C, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31 :3497-500.
Randall LL & Hardy SJ (2002) SecB, one small chaperone in the complex milieu of the cell. Cell Mol Life Sci 59: 1617-1623
Rizzitello AE, Harper JR, & Silhavy TJ (2001) Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli. J Bacferiol183: 6794-6800
Roe SM, Ali MM, Meyer P, Vaughan CK, Panaretou B, Piper PW, Prodromou C, & Pearl LH (2004) The Mechanism of Hsp9O regulation by the protein kinase-specific cochaperone p50(cdc37). Cell 11 6: 87-98
Rommelaere H, De Neve M, Melki R, Vandekerckhove J, & Ampe C (1999) The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins. Biochemistry 38: 3246-3257
Rommelaere H, De Neve M, Neirynck K, Peelaers D, Waterschoot D, Goethals M, Fraeyman N, Vandekerckhove J, & Ampe C (2001) Prefoldin recognition motifs in the nonhomologous proteins of the actin and tubulin families. J Biol Chem 276: 41 023-41 028
Rosendal KR, Wild K, Montoya G, & Sinning 1 (2003) Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication. Proc Natl Acad Sci U S A 100: 14701 -14706
Rudiger S, Germeroth L, Schneider-Mergener J, & Bukau B (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose- bound peptide libraries. EM50 J 16: 1501-1 507
Sambrook J, Fritsch EF, & Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY
Sauer FG, Barnhart M, Choudhury D, Knight SD, Waksman G, & Hultgren SJ (2000) Chaperone-assisted pilus assembly and bacterial attachment. Curr Opin Struct Biol10: 548-556
Sauer FG, Futterer K, Pinkner JS, Dodson KW, Hultgren SJ, & Waksman G (1999) Structural basis of chaperone function and pilus biogenesis. Science 285: 1058-1 061
Sauer FG, Pinkner JS, Waksman G, & Hultgren SJ (2002) Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation. Cell 1 11 : 543-551
Schaffitzel C, Oswald M, Berger I, lshikawa T, Abrahams JP, Koerten HK, Koning RI, & Ban N (2006) Structure of the E. coli signal recognition particle bound to a translating ribosome. Nature 444: 503-506
Schaffitzel E, Rudiger S, Bukau B, & Deuerling E (2001) Functional dissection of trigger factor and DnaK: interactions with nascent polypeptides and thermally denatured proteins. Biol Chem 382: 1235-1 243
Schlieker C, Weibezahn J, Patzelt HI Tessarz P, Strub C, Zeth K, Erbse A, Schneider-Mergener J, Chin JW, Schultz PG, Bukau B, & Mogk A (2004) Substrate recognition by the AAA+ chaperone ClpB. Nat Struct Mol Biol 11 : 607-61 5
Schubot FD, Jackson MW, Penrose KJ, Cherry S, Tropea JE, Plano GV, & Waugh DS (2005) Three-dimensional structure of a macromolecular assembly that regulates type Ill secretion in Yersinia pestis. J Mol Biol 346: 1147-1 161
Schulze-Gahmen U, Aono S, Chen S, Yokota H, Kim R, & Kim SH (2005) Structure of the hypothetical Mycoplasma protein MPN555 suggests a chaperone function. Acta Crystallogr D 5iol Crystallogr 61 : 1343-1 347
Shapova YA & Paetzel M (2007) Crystallographic analysis of Bacillus subtilis CsaA. Acta Ctystallogr D Biol Crystallogr 63: 478-485
Shiau AK, Harris SF, Southworth DR, & Agard DA (2006) Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell 127: 329-340
Siegert R, Leroux MR, Scheufler C, Hartl FU, & Moarefi 1 (2000) Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103: 621 -632
Spiess C, Meyer AS, Reissmann S, & Frydman J (2004) Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol14: 598-604
Spiess C, Miller EJ, McClellan AJ, & Frydman J (2006) Identification of the TRiClCCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins. Mol Cell 24: 25-37
Stebbins CE & Galan JE (2001) Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type Ill secretion. Nature 414: 77-81
Stephenson K (2005) Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review). Mol Membr Biol22: 17-28
Stirling PC, Bakhoum SF, Feigl AB, & Leroux MR (2006) Convergent evolution of clamp-like binding sites in diverse chaperones. Nat Struct Mol Biol 13: 865-870
Swairjo MA, Morales AJ, Wang CC, Ortiz AR, & Schimmel P (2000) Crystal structure of trbpl11: a structure-specific tRNA-binding protein. EM80 J 19: 6287-6298
Takeda K, Miyatake H, Yokota N, Matsuyama S, Tokuda H, & Miki K (2003) Crystal structures of bacterial lipoprotein localization factors, LolA and LolB. EM80 J 22: 3199-3209
Tang YC, Chang HC, Roeben A, Wischnewski D, Wischnewski N, Kerner MJ, Hartl FU, & Hayer-Hartl M (2006) Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein. Cell 125: 903-91 4
Thompson JD, Higgins DG, & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680
Tokuda H & Matsuyama S (2004) Sorting of lipoproteins to the outer membrane in E. coli. Biochim Biophys Acta 1694: IN1 -9
Tovchigrechko A & Vakser IA (2006) GRAMM-X public web server for protein- protein docking. Nucleic Acids Res 34: W 3 1 0-4
Tranier S, lobbi-Nivol C, Birck C, llbert M, Mortier-Barriere I, Mejean V, & Samama JP (2003) A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia. Structure 11: 165- 174
Ullers RS, Houben EN, Brunner J, Oudega B, Harms N, & Luirink J (2006) Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle. J Biol Chem 281: 13999- 14005
Vainberg IE, Lewis SA, Rommelaere HI Ampe C, Vandekerckhove J, Klein HL, & Cowan NJ (1998) Prefoldin, a chaperone that delivers unfolded proteins tc cytosolic chaperonin. Cell 93: 863-873
Valpuesta JM, Carrascosa JL, Willison KR (2005) Structure and Function of the Cytosolic Chaperonin CCT. In Protein Folding Handbook, Buchner J & Kiefhaber T (eds) pp 725-755. WILEY-VCH Verlag GmbH & Co.: KGaA, Weinheim
Vetsch M, Puorger C, Spirig TI Grauschopf U, Weber-Ban EU, & Glockshuber R (2004) Pilus chaperones represent a new type of protein-folding catalyst. Nature 431 : 329-333
Vogel M, Mayer MP, & Bukau B (2006) Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker. J Biol Chem 281 38705-3871 1
Walton TA & Sousa MC (2004) Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation Mol Cell 15: 367-374
Watanabe S, Matsuyama S, & Tokuda H (2006) Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli. J Bio Chem 281 : 3335-3342
Weibezahn J, Tessarz P, Schlieker C, Zahn R, Maglica Z, Lee S, Zentgraf H, Weber-Ban EU, Dougan DA, Tsai FT, Mogk A, & Bukau B (2004) Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell 119: 653-665
Xia D, Esser L, Singh SK, Guo F, & Maurizi MR (2004) Crystallographic investigation of peptide binding sites in the N-domain of the ClpA chaperone. J Struct Biol146: 166-1 79
Xu Z, Honvich AL, & Sigler PB (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388: 741 -750
Xu Z, Knafels JD, & Yoshino K (2000) Crystal structure of the bacterial protein export chaperone secB. Nat Struct Biol7: 1 172-1 177
Yamane K, Bunai K, & Kakeshita H (2004) Protein traffic for secretion and related machinery of Bacillus subtilis. Biosci Biotechnol Biochem 68: 2007- 2023
Yi L & Dalbey RE (2005) OxallAlb3lYidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review). Mol Membr Biol22: 101-111
Yip CK, Finlay BB, & Strynadka NC (2005) Structural characterization of a type Ill secretion system filament protein in complex with its chaperone. Nat Struct Mol Biol12: 75-81
Young JC, Agashe VR, Siegers K, & Hart1 FU (2004) Pathways of chaperone- mediated protein folding in the cytosol. Nat Rev Mol Cell Biol5: 781-791
Zhao R & Houry WA (2007) Molecular interaction network of the Hsp9O chaperone system. Adv Exp Med Biol594: 27-36
Zhou J & Xu Z (2005) The structural view of bacterial translocation-specific chaperone SecB: implications for function. Mol Microbiol 58: 349-357
Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM, Gottesman ME, & Hendrickson WA (1 996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272: 1606-1 614
Zolkiewski M (2006) A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases. Mol Microbiol 61 : 1094-1 100