protein networks / protein complexes

5. Lecture WS 2004/05

Bioinformatics III 1

Protein Networks / Protein Complexes

Protein networks could be defined in a number of ways

(1) Co-regulated expression of genes/proteins

(2) Proteins participating in the same metabolic pathways

(3) Proteins sharing substrates

(4) Proteins that are co-localized

(5) Proteins that form permanent supracomplexes = „protein machines“

(6) Proteins that bind eachother transiently

(signal transduction, bioenergetics ... )

In the next weeks, we will consider direct interactions (5) and (6).



Structural Techniques

Russell et al. Curr. Opin. Struct. Biol. 14, 313 (2004)



Potential pitfalls




Hybrid models: docking X-ray structures into EM maps




A biological cell: a large construction site?

Job office publishes lists (DNA) of people looking for jobs (protein). Managers from the personnel office (DNA-transcription factors) recruit (express) proteins.

Workers (proteins) need to get to their working places (localization).

During work they get energy from drinking beer (ATP).

In a biological cell there are many tasks that need to be executed in a timely and precise manner.

All steps depend on interaction of proteins with DNA or with other proteins!



1 Protein-Protein Complexes

It has been realized for quite some time that cells don‘t work by random

diffusion of proteins,

but require a delicate structural organization into large protein complexes.

Which complexes do you know?



RNA Polymerase II

RNA polymerase II is the

central enzyme of gene

expression and synthesizes all

messenger RNA in

eukaryotes.

Cramer et al., Science 288, 640 (2000)



RNA processing: splicesome

Structure of a cellular editor that "cuts and pastes" the first draft of RNA straight

after it is formed from its DNA template. It has two distinct, unequal halves

surrounding a tunnel. The larger part appears to contain proteins and the short

segments of RNA, while the smaller half is made up of proteins alone. On one

side, the tunnel opens up into a cavity, which the researchers think functions as

a holding space for the fragile RNA waiting to be processed in the tunnel itself.

Profs. Ruth and Joseph Sperlinghttp://www.weizmann.ac.il/



Protein synthesis: ribosome

The ribosome is a complex

subcellular particle composed of

protein and RNA. It is the site of

protein synthesis,

http://www.millerandlevine.com/chapter/12/cryo-em.html

Model of a ribosome with a

newly manufactured protein

(multicolored beads) exiting

on the right.



Signal recognition particle

40S small ribosomal subunit

(yellow) 60S large ribosomal

subunit (blue), P-site tRNA

(green), SRP (red).

Halic et al. Nature 427, 808 (2004)

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. SRP shows 3 main activities in the process of cotranslational targeting: first, it binds to signal sequences emerging from the translating ribosome; second, it pauses peptide elongation; and third, it promotes protein translocation by docking to the membrane-bound SRP receptor and transferring the ribosome nascent chain complex (RNC) to the protein-conducting channel.



Nuclear Pore ComplexA three-dimensional image of the

nuclear pore complex (NPC),

revealed by electron microscopy.

A-B The NPC in yeast.

Figure A shows the NPC seen

from the cytoplasm while figure B

displays a side view.

C-D The NPC in vertebrate

(Xenopus).

http://www.nobel.se/medicine/educational/dna/a/transport/ncp_em1.htmlThree-Dimensional Architecture of the Isolated Yeast Nuclear Pore Complex: Functional and Evolutionary Implications, Qing Yang, Michael P. Rout and Christopher W. Akey. Molecular Cell, 1:223-234, 1998

NPC is a 50-100 MDa protein assembly that

regulates and controls trafficking of

macromolecules through the nuclear envelope.

http://www.nobel.se/medicine/educational/dna/a/transport/ncp_em1.html

http://www.nobel.se/medicine/educational/dna/a/transport/ncp_em1.html



GroEL: a chaperone to assist misfolded proteins

Schematic Diagram of GroEL Functional States(a) Nonnative polypeptide substrate (wavy black line) binds to an open GroEL ring. (b) ATP binding to GroEL alters its conformation, weakens the binding of substrate, and permits the binding of GroES to the ATP-bound ring. (c) The substrate is released from its binding sites and trapped inside the cavity formed by GroES binding. (d) Following encapsulation, the substrate folds in the cavity and ATP is hydrolysed. (e) After hydrolysis in the upper, GroES-bound ring, ATP and a second nonnative polypeptide bind to the lower ring, discharging ligands from the upper ring and initiating new GroES binding to the lower ring (f) to form a new folding active complex on the lower ring and complete the cycle.

http://people.cryst.bbk.ac.uk/~ubcg16z/chaperone.html

Ransom et al., Cell 107, 869 (2001)



Arp2/3 complex

The seven-subunit Arp2/3 complex choreographs the formation of branched actin

networks at the leading edge of migrating cells.

(A) Model of actin filament branches mediated by Acanthamoeba Arp2/3 complex.

(D) Density representations of the models of actin-bound (green) and the free, WA-

activated (as shown in Fig. 1D, gray) Arp2/3 complex.

Volkmann et al., Science 293, 2456 (2001)



proteasome

The proteasome is the central

enzyme of non-lysosomal protein

degradation. It is involved in the

degradation of misfolded proteins

as well as in the degradation and

processing of short lived regulatory

proteins.The 20S Proteasome

degrades completely unfoleded

proteins into peptides with a

narrow length distribution of 7 to

13 amino acids.

http://www.biochem.mpg.de/xray/projects/hubome/images/rpr.gifLöwe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W. and Huber, R. (1995). Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533-539.

http://www.biochem.mpg.de/xray/projects/hubome/images/rpr.gif

http://www.biochem.mpg.de/xray/projects/hubome/images/rpr.gif



Energy conversion: Photosynthetic Unit

Structure suggested by

force field based

molecular docking.

http://www.ks.uiuc.edu/Research/vmd/gallery

Other large complexes:

- Apoptosome-Thermosome- Transcriptome



icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine

Model for active-site coupling in the E1E2 complex. 3 E1 tetramers (purple) are shown located above the corresponding trimer of E2 catalytic domains in the icosahedral core. Three full-length E2 molecules are shown, colored red, green and yellow. The lipoyl domain of each E2 molecule shuttles between the active sites of E1 and those of E2. The lipoyl domain of the red E2 is shown attached to an E1 active site. The yellow and green lipoyl domains of the other E2 molecules are shown in intermediate positions in the annular region between the core and the outer E1 layer. Selected E1 and E2 active sites are shown as white ovals, although the lipoyl domain can reach additional sites in the complex.

Milne et al., EMBO J. 21, 5587 (2002)



Apoptosome(A) Top view of the apoptosome along the 7-fold

symmetry axis.

(B) Details of the spoke.

(C) A side view of the apoptosome reveals the

unusual axial ratio of this particle. The scale bar is

100 Å.

(D) An oblique bottom view shows the puckered

shape of the particle. The arms are bent at an

elbow (see asterisk) located proximal to the hub.

Acehan et al. Mol. Cell 9, 423 (2002)

Apoptosis is the dominant form of programmed cell death during embryonic development and normal tissue turnover. In addition, apoptosis is upregulated in diseases such as AIDS, and neurodegenerative disorders, while it is downregulated in certain cancers. In apoptosis, death signals are transduced by biochemical pathways to activate caspases, a group of proteases that utilize cysteine at their active sites to cleave specific proteins at aspartate residues. The proteolysis of these critical proteins then initiates cellular events that include chromatin degradation into nucleosomes and organelle destruction. These steps prepare apoptotic cells for phagocytosis and result in the efficient recycling of biochemical resources.In many cases, apoptotic signals are transmitted to mitochondria, which act as integrators of cell death because both effector and regulatory molecules converge at this organelle. Apoptosis mediated by mitochondria requires the release of cytochrome c into the cytosol through a process that may involve the formation of specific pores or rupture of the outer membrane. Cytochrome c binds to Apaf-1 and in the presence of dATP/ATP promotes assembly of the apoptosome. This large protein complex then binds and activates procaspase-9.



Future?

Structural genomics (X-ray) may soon generate enough templates of individal

folds.

Structural genomics may be expanded to protein complexes.

Interactions between proteins of the same fold tend to be similar when the

sequence identity is above approximately 30% (Aloy et al.).

Hybrid modelling of X-ray/EM will not be able to answer all questions- problem of induced fit- transient complexes cannot be addressed by these techniques

Essential to combine large variety of hybrid + complementary methods




2 Information on protein-protein networks



2. Yeast 2-Hybrid Screen

Data on protein-protein interactions fromYeast 2-Hybrid Screen.

One role of bioinformatics is tosort the data.



Protein cluster in yeast

Schwikowski, Uetz, Fields, Nature Biotech. 18, 1257 (2001)

Cluster-algorithm generates one largecluster for proteins interacting with eachother based on binding data of yeast proteins.



Annotation of function


After functional annotation:connect clusters ofinteracting proteins.



Annotation of localization




3 Systematic identification of protein complexes



Systematic identication of large protein complexesYeast 2-Hybrid-method can only identify binary complexes.

Cellzome company: attach additional protein P to particular protein Pi ,

P binds to matrix of purification column.

yields Pi and proteins Pk bound to Pi .

Gavin et al. Nature 415, 141 (2002)

Identify proteinsby mass spectro-metry (MALDI-TOF).



Analyis of protein complexes in yeast (S. cerevisae)


Identify proteins by

scanning yeast protein

database for protein

composed of fragments

of suitable mass.

Here, the identified

proteins are listed

according to their

localization (a).

(b) lists the number of

proteins per complex.



Validation of methodology


Check of the method: can the same complex be obtained for differentchoice of attachment point(tag protein attached to different coponents of complex)? Yes (see gel).

Method allows to identify components of complex, not the binding interfaces.

Better for identification of interfaces:Yeast 2-hybrid screen (binary interactions).

3D models of complexes are importantto develop inhibitors.

- theoretical methods (docking) - electron tomography



Network of protein complexes?


Service function of Bioinformatics: catalog such data and prepare for analysis ...

allowing to formulate new models and concepts (biology!).

If results are very important don‘t wait for some biologist to interpret your data. You may want to get the credit yourself.

Modularity = Formation of separated Islands ??



Experiment

Start from 232 purified complexes from TAP strategy.

Select 102 that gave samples most promising for EM from analysis of gels and

protein concentrations.

Take EM images.

Theory

Make list of components.

Assign known structures of individual proteins.

Assign templates of complexes-If complex structure available for this pair- if complex structure available for homologous protein- if complex structure available for structurally similar protein (SCOP)

4 Aim: generate structures of protein complexes

Bettina Böttcher (EM)Rob Russell (Bioinformatics)



How transferable are interactions?interaction similariy (iRMSD) vs. %

sequence identity for all the available

pairs of interacting domains with

known 3D structure.

Curve shows 80% percentile (i.e. 80%

of the data lies below the curve), and

points below the line (iRMSD = 10 Å)

are similar in interaction. Aloy et al. Science, 303, 2026 (2004)



Bioinformatics Strategy

Illustration of the methods and concepts

used. How predictions are made within

complexes (circles) and between them

(cross-talk). Bottom right shows two

binary interactions combined into a three-

component model

Aloy et al. Science, 303, 2026 (2004)



3SOM algorithm: vector-based circumference superimposition

A 2D variant of the 3D vector-based surface

superimposition that is central to the 3SOM

algorithm. For each tested voxel a on the

circumference of the target, a vector va is

calculated that approximates the normal vector

orthogonal to the tangent line in a and with origin

in a. Vector va is superimposed on each vector vb

that is associated with a voxel b on the

circumference of the template. The goodness-of-

fit of the transformation in question is assessed by

measuring the circumference overlap, the fraction

of target circumference voxels that is projected

onto (or near) the template circumference

(triangles). In 3D, a rotational degree of freedom

is left around the superimposed vectors, which is

sampled in rotational steps of 9°.

Ceulemans, Russell J. Mol. Biol., 338, 783 (2004)



Successful models of yeast complexes

(A) Exosome model on PNPase fit into

EM map.

(B) RNA polymerase II with RPB4

(green)/RPB7 (red) built on

Methanococcus jannaschii equivalents,

and SPT5/pol II (cyan) built with IF5A.

(C and D) Views of CCT (gold) and

phosphoducin 2/VID27 (red) fit into EM

map.

(E) Micrograph of POP complex, with

particle types highlighted.

(F) Ski complex built by combination of

two complexes.




Cross talk between complexes

(Top) Triangles show

components with at least one

modelable structure and

interaction; squares, structure

only; circles, others.

Lines show predicted

interactions: thick lines imply a

conserved interaction interface;

red, those supported by

experiment.

(Bottom) Expanded view of

cross-talk between transcription

complexes built on by a

combination of two complexes.




SummaryA combination of 3D structure and protein-interaction data can already provide a partial view of complex cellular structures.

The structure-based network derived from cross-talk between complexes provides a more realistic picture than those derived blindly from interaction data, because it suggests molecular details for how they are mediated.

Of course, the picture is still far from complete and there are numerous new challenges.

The structure-based network derived here provides a useful initial framework for further studies. Its beauty is that the whole is greater than the sum of its parts: Each new structure can help to understand multiple interactions.

The complex predictions and the associated network will thus improve exponentially as the numbers of structures and interactions increase, providing an ever more complete molecular anatomy of the cell.


protein networks / protein complexes

Documents

protein machines6 proteins

proteinprotein complexesit

express proteins

interaction of proteins

workers proteins

jobs protein

colocalized5 proteins

large protein complexes