The Importance of Non-conserved Regions in Protein Remodeling by the E. coli Molecular Chaperone,

ClpB

Zakiya Qualls

Background • Molecular chaperones are a family of proteins that

aid in the prevention of protein misfolding and aggregation.

• Protein aggregation often occurs following extreme environmental stress, for example heat stress, which can lead to the loss of protein activity and cell death.

• Protein aggregates, called amyloids are involved in diseases, including neurological disorders such as Alzheimer’s, Huntington’s, and Parkinson’s.

ClpB

• ClpB Is a molecular chaperone that is required for growth at high temperatures (thermotolerance).

• It belongs to the AAA+ (ATPases associated with various cellular activities) superfamily of ATPases.

• Both in vivo and in vitro, ClpB is required for protein disaggregation and reactivation.

• ClpB works with the DnaK chaperone system to disaggregate proteins.

• Yeast Hsp104, plant Hsp101, and mitochondrial Hsp78 are the homologs of ClpB.

ClpBTop view of hexamer

ATP(yellow)

Channel

Middle domain(red)

ATP

Channel

NBD-1

NBD-2

Side view of hexamer

• ClpB is a hexamer containing a central channel involved in protein unfolding and translocation.

• Each monomer is comprised of an N-domain and two nucleotide-binding domains (NBD) as well as a unique coiled-coil middle (M) domain.

Model of E. coli ClpB based on T. thermophilus structure.

Structure: Lee et al., Cell, 2003Hexamer model: Diemand & Lupas,

J. Struct. Biol., 2006

Middle domain(red) N-terminal

domain(green)

Goals Question: What are the roles of the N-terminal domain

and the M-domain in ClpB chaperone activity?

To determine if mutations in the N-terminal domain

and the M-domain of ClpB affect its protein

remodeling abilities.

1. Construct N-domain and M-domain mutants.2. Purify active ClpB mutant proteins. 3. Examine the unfolding capabilities of the ClpB mutant

proteins compared with wild-type ClpB. 4. Examine GFP disaggregation by ClpB mutant proteins.

ClpB N-domain and M-domain Mutants

M-2

M-3

M-1

N-1

N-2• The N- and M-domains have low sequence homology among species.

• An N-terminal deletion alters the function of ClpB both in vivo and in vitro.

• The M-domain is unique to ClpB and its homologs and required for chaperone activity.

Methods Mutant Construction:

1. Selected sites for mutagenesis2. Designed primers3. Mutagenesis of ClpB gene (QuickChange II Site-

Directed Mutagenesis Kit)4. Transformation of plasmids (DH5 cells)5. Plasmid Prep (QIA prep Spin Miniprep Kit)6. Confirmed mutations by sequencing

Protein Preparation:1. Transformation of mutant plasmid into BL21 cells) 2. Growth and induction 3. Low speed & high speed spin4. Column Chromatography

Cell lysate of ClpB M-3 mutant SDS-PAGE

Induced with IPTG Uninduced

P S P S1ul 2.5ul 7.5ul2ul 8ul 15ul

High Speed

Low Speed

ClpB

Protein Purification - Column Chromatography Q-sepharose

Separation by charge S200

Separation by size and shape

17 188 1512 1410 11 16139 2316 222119 20

ClpB(M-2)

M.W.

110 kDa80 kDa

60 kDa

50 kDa40 kDa

171512 1410 11 16139 14110 kDa80 kDa

60 kDa

50 kDa40 kDa

ClpB(M-3)

17 1815 16 222119 20

29 30 32 343331 35 36 37 38

ClpB(M-1)

110 kDa80 kDa

60 kDa

50 kDa40 kDa

8 9 10 11 12 13 14 15 16

17 1815 16 22 23 242119 2011 12 13 14 15 16 17 18

ClpB(N-1)

110 kDa80 kDa

60 kDa

50 kDa40 kDa

Unfolding of tagged-GFP by ClpB

Native fluorescent tagged-GFP(Green Fluorescent Protein)

Incubate with ClpB andnucleotide

Unfolded non-fluorescent GFP

Measure decrease in fluorescence

• The M-domain mutants are not significantly different than wild-type ClpB.

• The N-terminal domain mutants N-1 and N-2 are defective compared to wild-type ClpB.

NoClpB

WT

M-2M-1M-3

0.75

0.8

0.85

0.9

0.95

1

1.05

0 10 20 30 40 50 60Time (min)

Fluorescence Intensity

(A.U.)

M-domain mutants

0.45

0.55

0.65

0.75

0.85

0.95

1.05

0 10 20 30 40 50 60

Time (min)

Fluorescence Intensity

(A.U.)

No ClpB

WTN-2

N-1

N-terminal domain mutants

Measure increase in fluorescence

Incubate with ClpBand nucleotide

Heat-aggregatednon-fluorescent GFP Refolded GFP

Dissagregation of GFP by ClpB

• The three M-domain mutants are defective in protein disaggregation compared to wild-type ClpB.

• N-1 and N-2 have similar disaggregation activity compared to wild-type ClpB

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Time (min)

Fluorescence Intensity

(A.U.)

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Time (min)

Fluorescence Intensity

(A.U.)

NoClpB

N-1WTN-2

No ClpB

WT

M-2

M-1M-3

M-domain mutants N-terminal domain mutants

Measure increase in fluorescence

Incubate with ClpB + DnaK chaperone system

and nucleotide

Heat-aggregatednon-fluorescent GFP Refolded GFP

Disaggregation of GFP by ClpB with DnaK Chaperone System

• The M-domain mutants are defective compared to wild-type ClpB in disaggregation activity with the DnaK chaperone system.

No Chaperone

WT

M-2M-1

M-3

DnaK chaperone system alone

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60

Time (min)

Fluorescence Intensity

(A.U.)

M-domain mutants

Conclusions• ClpB mutants in the N -terminal domain have decreased protein unfolding activity

compared to wild-type, but have disaggregation activity similar to wild-type in the absence of the DnaK chaperone system.

• ClpB M-domain mutants possess protein unfolding activity similar to wild-type, but have reduced disaggregation activity alone and in the presence of the DnaK system.

• The M-domain may be important for protein disaggregation by ClpB in the presence and

absence of the DnaK chaperone system.

• This and further research will help understand how molecular chaperones interact with other proteins and how they may be vital in fighting several neurological disorders.

Acknowledgements • Dr. Sue Wickner• Shannon Doyle• Danielle Johnston • Jodi Camberg• Joel Hoskins• Marika Miot• Olivier Genest• NIH Summer Internship Program in Biomedical

Research• The Howard University COR Honors Research

Program