Roles of individual domains and conserved motifs of the ... · 2003-03-06 · Axel Mogk 1,2,3, Christian Schlieker 1,2, Christine Strub 1,2, Wolfgang Rist 1,2, Jimena Weibezahn 1,2

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB

in oligomerization, ATP-hydrolysis and chaperone activity

Axel Mogk 1,2,3, Christian Schlieker 1,2, Christine Strub 1,2, Wolfgang Rist 1,2, Jimena

Weibezahn 1,2 and Bernd Bukau 1,2,3

1 Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-

Herder-Str. 7, 79104 Freiburg

2 present address: Zentrum für Molekulare Biologie Heidelberg (ZMBH), Universität

Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany

3 Corresponding author

email: [email protected]

[email protected]

Running title: Structure function analysis of ClpB

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 6, 2003 as Manuscript M209686200 by guest on O

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Summary

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates

the resolublization of aggregated proteins in cooperation with the DnaK chaperone

system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is

composed of an N-terminal domain and two AAA domains which are separated by a

“linker” region. Here we present a detailed structure-function analysis of ClpB,

dissecting the individual roles of ClpB domains and conserved motifs in

oligomerization, ATP hydrolysis and chaperone activity. Our results show that ClpB

oligomerization is strictly dependent on the presence of the C-terminal domain of the

second AAA domain, while ATP-binding to the first AAA domains stabilised the

ClpB oligomer. Analysis of mutants of conserved residues in Walker A, B and sensor

2 motifs revealed that both AAA domains contribute to the basal ATPase activity of

ClpB and communicate in a complex manner. Chaperone activity strictly depends on

ClpB oligomerization and the presence of a residual ATPase-activity. The N-domain

is dispensible for oligomerization and for the disaggregating activity in vitro and in

vivo. In contrast the presence of the “linker” region, while not involved in

oligomerization, is essential for ClpB chaperone activity.

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Introduction

The Escherichia coli chaperone ClpB belongs to the ring-forming Clp/Hsp100

proteins. Clp/Hsp100 proteins can be classified into two distinct subfamilies. Class I

proteins (ClpA and ClpB in E. coli) are composed of two highly conserved nucleotide

binding domains (termed ATP-1 and ATP-2,) while class II proteins (ClpX, HslU as

representatives of E. coli) proteins contain only a single NBD (homologous to ATP-2)

(1). Sequence analysis of the NBD´s revealed a significant sequence homology

between Clp/Hsp100 and AAA proteins (ATPase associated with a variety of cellular

activities) and consequently a new AAA+ superfamily, representing both protein

classes, was proposed (2). The structural basis of this superfamily was confirmed by

determination of the first Clp/Hsp100 protein structure, HslU, which showed

significant similarity to the AAA proteins N-ethylmaleimide-sensitive fusion protein

(NSF) and p97 (3,4). The recently solved crystal structure of the first nucleotide

binding domain of ClpB also demonstrated the close structural relationship between

Clp/Hsp100 and AAA proteins (5). The conserved AAA domain (also referred to as

AAA module) is made up of two domains, a core region which forms the nucleotide

binding pocket, containing the classical Walker A and B motifs, and a C-terminal α-

helical domain (C-domain). The ATP binding pocket is located at the interface of

neighbouring subunits in the oligomer. The C-domain contacts its own core ATPase

domain and that of adjacent subunits and is involved in nucleotide binding and

hexamerization in HslU (3,4). Besides sensing the nucleotide status of the core

ATPase domain, C-terminal domains of the second AAA domain have been also

proposed to mediate substrate interaction and were therefore termed the sensor- and

substrate discrimination (SSD) domains (6).


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In addition Hsp100/Clp proteins contain variable regions at their N-terminus. ClpA

and ClpB have homologous N-domains of about 150 residues that consist of two

sequence repeats and form an independent structural domain still of unknown

function (7). ClpX possesses a Zn-binding domain at the N-terminus (8), whereas

HslU lacks an N-terminal domain but rather contains an extra domain (the I-domain)

inserted into the AAA domain (3,4). The most striking difference between members

within the Class I sub-family is the presence/absence of a region, which is proposed to

link the two AAA domains ATP-1 and ATP-2. The presence of this variable “linker”

region serves as a criteria for classification of Hsp100 proteins: the “linker” is longest

in ClpB (approx. 140 residues), but is absent in ClpA (see Figure 1) (1).

ClpB is unique among the Hsp100/Clp proteins since it does not associate with a

proteolytic partner protein. Recently, an essential docking site of the peptidase ClpP

was identified within ClpX (9). The signature motif (LIV-G-FL) is conserved in all

other ClpP interacting proteins like ClpA, but is missing in ClpB, thereby explaining

why ClpB acts independently of peptidases. Instead ClpB mediates the

resolubilization of aggregated proteins in cooperation with the DnaK chaperone

system (10-13). The mechanism of the disaggregation reaction and the basis of

ClpB/DnaK cooperation are still not understood.

Here we report a structure-function analysis of ClpB which is aimed at identifying the

roles of individual domains and conserved motifs in the disaggregation process and

the coupling of the ATPase-cycle with the chaperone activity. Constructed ClpB

variants were characterized with respect to their structural integrity, as determined by

oligomerization studies and partial proteolysis. Additionally the ATPase and

chaperone activities of all constructs were tested.


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Experimental procedures

Strains and plasmids

E. coli strains used were derivatives of MC4100 (araD139 D(argF-lac)U169 rpsL150

relA1 flbB5301 deoC1 ptsF25 rbsR). clpB mutant allele was introduced by P1

transduction into MC4100 background to generate strains BB4561 (∆clpB::kan). The

E. coli clpB gene was cloned by PCR into the pUHE21 expression vector and verified

by DNA sequencing. Mutants and deletion variants were generated by using standard

PCR techniques. ClpB ∆410-532 was constructed by replacing the entire “linker”

region with a SpeI site, leading to the insertion of two amino acids (T,S) at the

deletion site. Reinsertion of the linker was obtained by PCR-amplification of the

corresponding region (aa 410-530) and addition of a SpeI site 5´and a NheI site 3´ to

the linker fragment. This fragment was digested with NheI/SpeI and inserted into the

SpeI site of the ∆410-532 construct, leading to the insertion of two amino acids (T,S

or A,S) at each domain boundary (ClpB ∆LBL). Each mutagenesis was confirmed by

DNA sequencing.

Proteins

Wild type and mutant ClpB were purified as described after overproduction in

∆clpB::kan cells (11). Purifications of DnaK, DnaJ and GrpE were according to

published protocols (14). Pyruvate kinase and α-casein were purchased from Sigma,

Malate Dehydrogenase (MDH) from pig heart muscle and firefly Luciferase from

Roche. Protein concentrations were determined with the Bio-Rad Bradford assay

using BSA as standard. Protein concentrations refer to the protomer.


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Tryptophan fluorescence

Measurements of the intrinsic tryptophan fluorescence of ClpB were performed on a

Perkin-Elmer LS50B spectrofluorimeter. The emission spectra of tryptophan

fluorescence of ClpB (0,5 µM) in the absence of nucleotide or the presence of 2 mM

ATP/ADP were recorded at 30°C in buffer A (50 mM Tris, pH 7.5; 150 mM KCl; 20

mM MgCl2; 2 mM DTT) between 300 and 400 nm at a fixed excitation wavelength of

290 nm.

ATPase-activity assay

ATP hydrolysis rates under steady-state conditions were determined as described

(14). Reactions were performed at 30°C in buffer A (50 mM Tris, pH 7.5; 150 mM

KCl; 20 mM MgCl2; 2 mM DTT) containing 0,5 µM ClpB (wild type or derivatives),

2 mM ATP and [α-32P]ATP (0.1 µCi, Amersham). ATPase activities were also

determined in the presence of 0,25 mg/ml α-casein or 100 mM ammonium sulfate.

Hydrolysis was quantified by using the program MACBAS version 2.5 (Fuji) and

rates of ATP hydrolysis were determined by using the programm GRAFIT version 3.0

(Erithacus software).

Size exclusion chromatography

Size exclusion chromatography was performed at room temperature in buffer A

containing 5% (v/v) glycerol. Nucleotide-dependent oligomerization was followed in

the presence of 2 mM ATP or ADP in the running buffer. 10 µM ClpB was incubated

in buffer A in the absence or presence of nucleotides (2 mM ATP/ADP) for 5 min at

room temperature, followed by injection into the HPLC system (Perkin-Elmer)


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connected to a SEC 400 column (Biorad). Chromatographic steps were performed

with a flow rate of 0.8 ml/min.

Cross-linking assays

All ClpB variants were dialyzed against buffer B (50 mM HEPES pH 7.5; 150 mM

KCl; 20 mM MgCl2; 2 mM DTT). 1 µM ClpB was incubated at 30°C in the absence

or presence of nucleotides (2 mM ATP or ADP) for 5 min. Cross-linking reactions

were started by addition of 0,1% glutaraldehyde and incubated for 10 min further.

Reactions were stopped by addition of 1 M Tris, pH 7,5 and cross-linking products

were analysed by SDS-PAGE (4-15%) followed by silver staining.

Partial proteolysis and identification of cleavage products

1 µM ClpB (wild type or derivative) was incubated at 30°C in buffer A without DTT

for 5 min in the absence or presence of nucleotide (2 mM ATP, ADP, ATPγS).

Proteolysis was initiated upon addition of 0,2 µg/ml thermolysin or subtilisin and

generated cleavage products were analysed by SDS-PAGE (15%) and silver staining.

Kinetic analysis of the degradation reaction revealed the occurrence of stable

fragments after 30-60 min incubation time. Idendity of cleavage products was

determined by N-terminal sequencing (TopLab) and mass spectrometry. For mass

spectrometry analysis bands were excised from 1-D Coomassie silver stained SDS-

polyacrylamide gels and in-gel-digested with trypsin as described (15). Tryptic

peptides were analyzed by nanoelectrospray tandem mass spectrometry as described

(16) using a QSTART M Pulsar (MDS Sciex, Toronto, Canada) equipped with a

nanoES ion source (MDS Proteomics, Odense, Denmark). Sequence searches were

performed with the Protein and Peptide Software Suite (MDS Proteomics).


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In vitro activity assays

2 µM MDH was denatured in buffer A for 30 min at 47°C. Turbidity of 1 µM

aggregated MDH was measured at 30°C at an excitation and emission wavelength of

550 nm (Perkin-Elmer luminescence spectrometer LS50B). Decrease of light

scattering was followed upon addition of 1,5 µM ClpB (wild type or derivative), the

DnaK chaperone system (1 µM DnaK; 0,2 µM DnaJ; 0,1 µM GrpE) and an ATP

regenerating system (3 mM phosphoenolpyruvate; 20 ng/ml pyruvate kinase; 2 mM

ATP). Disaggregation rates were derived from the linear decrease of turbidity

between 5 and 30 min.

0,2 µM Luciferase was denatured in buffer A for 15 min at 43°C. Refolding of 0,1

µM aggregated luciferase was initiated by addition of 1 µM ClpB (wild type or

derivative), the DnaK system (0,5 µM DnaK; 0,1 µM DnaJ; 0,05 µM GrpE) and an

ATP regenerating system. Luciferase activities were determined as described and

refolding rates were calculated from the linear increase of luciferase activities

between 15 and 90 min.

In vivo activity assays

Survival of E. coli cells after exposure to lethal temperature was determined by

calculating the plating efficiency. Cells were grown in LB medium to mid-exponential

growth phase at 30°C followed by incubation at 50° for the indicated time. Serial

dilutions (10-3 to 10-7) of cells were prepared in LB medium and spotted onto LB

plates. Plates were incubated for 24 h at 30°C and colony numbers were determined

afterwards. Defects in protein disaggregation in ∆clpB::kan mutant cells were

followed by isolation of aggregated proteins as described (17).


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Results

Design of ClpB mutants and deletions variants

ClpB is proposed to consist of a N-terminal domain, two AAA domains (ATP-1,

ATP-2), which are separated by a “linker” region, and the C-terminal SSD (as

illustrated in figure 1). In order to probe the proposed domain organization of ClpB

several deletion variants were constructed, based on sequence and secondary structure

analysis of ClpB and its comparison with AAA+ proteins of known structure. Due to

the presence of an internal start codon two versions of E. coli ClpB exist in vivo: a

fullength protein (1-857) and a N-terminally truncated variant (149-857). Both

versions have been demonstrated to form mixed oligomers (18). In order to work with

uniform proteins we decided to mutate the internal start codon of wild type ClpB,

leading to the production of fullength ClpB only. This ClpB derivative exhibited in

vitro and in vivo chaperone activity, indistinguishable from ClpB expressed from the

wild type gene (data not shown). The corresponding construct was used as basis for

the construction of all ClpB derivatives.

The border between the N-domain and the first AAA domain is often characterized by

the presence of an internal start codon within the clpB gene (V149). Sequence

analysis of Hsp100 N-domains and secondary structure prediction of 15 different

ClpB proteins, revealed that the ClpB N-domain is built up of residues 1-144 (7) (data

not shown). We therefore decided to use M143 as start site for ClpB ∆N (143-857)

version. The C-terminal SSD was removed in the ClpB ∆SSD variant (1-758). AAA

domains were suggested to be separated by the insertion of the “linker” region within

ClpB. Recent sequence and structural analysis of AAA+ proteins revealed that the

linker region potentially interrupts the first AAA domain instead of separating both


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AAA modules (2). Several ClpB deletion variants were constructed to test this

possibility: ClpB 1-409, ClpB 551-857 and ClpB 1-567.

Several conserved motifs and residues are proposed to be involved in ATP binding

and hydrolysis by AAA proteins. The highly conserved Lys residues of the Walker A

motif (K212; K611 of ClpB) contact the β and γ nucleotide`s phosphate groups in the

first and second AAA domain, respectively (19). The conserved Glu residues of the

Walker B motif (E279; E678 of ClpB) are proposed to represent the catalytic base for

ATP hydrolysis in the first and second AAA domain, respectively. AAA+ proteins

additionally contain a conserved Arg residue, termed Sensor 2 (2). Sensor 2 lies in the

C-terminal domain of each AAA module and contacts the γ phosphate of bound ATP.

This Arg residue is part of an invariant GAR motif (813GAR815 in ClpB) within the

SSD domain of AAA+ (Hsp100) proteins. It is proposed that the conserved arginine

can sense the nucleotide status and mediate conformational changes of the C-terminal

domain relative to the core domain during ATP hydrolysis (19,20).

The formation of hexameric rings in AAA+ proteins brings residues of adjacent

protomers into close proximity to ATP, bound to a neighbouring subunit. Such

interactions could provide basis for an intermolecular catalytic mechanism, resulting

in cooperative ATP hydrolysis within the oligomer. Previous studies on the AAA

protein FtsH have demonstrated that Arg residues (R332; R756 in ClpB) can

potentially act as “Arg fingers” by contacting the ATP, bound to a neighboured

subunit (21). All described motifs, involved in ATP binding and hydrolysis, were

subjected to alanine mutagenesis, as illustrated in figure 1. ClpB mutants and deletion

variants were purified and characterized with respect to oligomerization, ATP

hydrolysis and chaperone activity.


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Oligomerization of ClpB variants

ClpB proteins form hexameric ring structures in the presence of ATP (22).

Oligomerization of ClpB mutants and deletion variants was followed by size

exclusion chromatography in the absence or presence of ATP and ADP. Consistent

with published data, ClpB is only capable of hexamerization in the presence of ATP

(Figure 2A). In the absence of nucleotide, ClpB eluted predominantly as a dimer,

while ADP addition led to the formation of trimers and/or tetramers. Analyis of ClpB

deletion variants revealed that isolated AAA domains (1-409; 1-567; 551-857) cannot

oligomerize. In agreement with previous data (23) hexamerization is strictly

dependent on the presence of the SSD domain, while the N-domain and the linker

region are dispensable (Table 1). Characterization of ClpB point mutants revealed,

that besides the C-terminal SSD domain the first ATPase domain also contributes to

oligomerization: mutations in the Walker A motif (K212A) or the “Arg finger”

(R332A) of the first AAA domain abolished hexamerization, while corresponding

mutations in the second AAA module (K611A; R756A) domain had no effect (Figure

2B, Table 1). Finally, all Walker B mutants in the two AAA modules (E279A;

E678A; E279A/E678A), as well as the Sensor 2 mutant (813AAA815), did not

exhibit oligomerization defects.

In an additional approach oligomerization characteristics were studied by

glutaraldehyde cross-linking. Fast cross-linking of ClpB monomers to oligomeric

species were obtained within 10 min in the absence of nucleotide, indicating that

ClpB assembly can in principle also occur without nucleotide. Since such nucleotide-

independent hexamerization was not observed during gelfiltration runs, the formed

oligomers seem to be unstable in the absence of ATP.


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Kinetic analyis of the cross-linking reaction revealed that a ladder of cross-linking

products preceeded the formation of the fully cross-linked oligomer (data not shown).

The presence of ATP or ADP accelerated the cross-linking reaction and also slightly

changed the size of the fully cross-linked oligomer. While the ladder of cross-link

products could be followed up to a heptamer in the absence of nucleotide, addition of

ATP and ADP resulted predominantly in cross-linking to hexameric and pentameric

species, respectively (Figure 3A). The existence of heptameric ClpB rings have also

been shown by Chung and co-workers (18) using electron microscopy.

Cross-linking studies of ClpB mutants and deletion variants confirmed the findings

obtained by gelfiltration analysis, however, qualitative differences with respect to the

oligomerization deficiencies were revealed. Isolated AAA domains (1-409; 551-857)

stayed as monomers in presence of glutaraldehyde, whereas in case of the ClpB ∆SSD

variant (1-758) and a longer version of the first AAA module (1-567) some dimeric

and trimeric species were observed (Figure 3B). Mutating the Walker A motif

(K212A) or the “Arg finger” (R332A) of the first AAA domain resulted in the

formation of mixtures of oligomeric species, ranging from monomers to tetramers in

case of K212A, and monomers to hexamers for R332A (Figure 3A, Table 1).

Interestingly, the observed oligomerization defects were nucleotide-independent,

indicating that the mutated residues are also important for subunit interactions within

the oligomer in the absence of ATP. The involvement of these charged residues in

ClpB assembly can also explain the observed salt-sensitivity of ClpB and Hsp104

oligomerization (24,25). All other ClpB variants exhibited cross-linking

characteristics indistinguishable from wild type ClpB with exception of ClpB ∆410-

532 which misses the “linker” region. This variant exhibited only a ladder of cross-

linking products in the absence of nucleotide and required ATP for full


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oligomerization. The observed assembly defect was, however, not primarily caused

by the absence of the “linker” region, but rather by the introduction of additional

amino acids at each boundary of the “linker” segment, resulting from the construction

strategy of this deletion variant (see experimental procedures). Thus a control

construct (termed ClpB ∆LBL), carrying the same additional amino acids and the

reinserted “linker” region, also exhibited the same oligomerization defects as ClpB

∆410-532 (data not shown), showing that the “linker” is not essential for

oligomerization.

Finally in a complementary approach we examined the intrinsic fluorescence of ClpB

as a potential tool to monitor the oligomeric state. ClpB contains 2 tryptophan

residues (W462, W543). We determined whether tryptophan fluorescence changes in

a nucleotide dependent fashion. In the absence of nucleotide ClpB exhibited a

fluorescence emission maximum at 350.5 nm. Addition of ATP or ADP caused a

blue-shift in the fluorescence maximum to 346.5 nm and a stronger decline of

fluorescence intensity beyond the maximum was observed (Figure 4A). This change

was more pronounced in the presence of ATP compared to ADP. Several findings

suggest that the observed changes in fluorescence are caused by oligomerization of

ClpB. Firstly the blue shift was also observed when ClpB was incubated in low salt

buffer in the absence of nucleotide (Figure 4). Such buffer conditions have been

shown for several ClpB homologues to stabilize the oligomer in the absence of

nucleotide (Schlee et al., 2001; Hattendorf and Lindquist, 2002). Consistently, the

ATP-dependent blue shift was reverted under high ionic strength conditions (addition

of 100 mM (NH4)2S04 or 300 mM NH4Cl), conditions which have been shown to

inhibit ClpB activity (10). Deoligomerization of ClpB in the additional presence of

100 mM (NH4)S04 or 300 mM NH4Cl was also observed in cross-linking experiments.


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Such high salt conditions resulted in a strong reduction of ClpB cross-linking

efficiency in the absence or presence of ATP (Figure 4B, data not shown).

Secondly, the observed changes in fluorescence became also nucleotide-independent

with increasing ClpB concentrations; a complete blue-shift was revealed in presence

of 4 µM ClpB (data not shown). Finally the analysis of ClpB mutants and deletion

variants revealed a direct correlation between the ability to form hexamers and the

observed changes in tryptophan fluorescence: the ATP-dependent blue-shift was not

or only partially observed for ClpB variants with oligomerization defects (Table 1).

In order to dissect the individual contributions of the two tryptophan residues to the

changes in the fluorescence spectrum, a ClpB variant (W543F) with only a single

tryptophan residue (W462) was constructed. While this variant was not affected in

oligomerization and exhibited full chaperone activity in vitro, nucleotide-dependent

changes in ClpB fluorescence were no longer observed (data not shown). We

conclude that conformational changes in the close vicinity of W543, driven by

oligomerization, must be responsible for the observed blue shift in tryptophan

fluorescence of ClpB.

Nucleotide-dependent conformational changes within ClpB revealed by partial

proteolysis

The ability of Hsp100 mutants to form oligomers is commonly used as a criteria for

their structural integrity. We additionally looked for conformational changes of wild

type ClpB in response to nucleotides and checked whether such structural

rearrangements are preserved in the constructed ClpB variants. ClpB was subjected to

limited proteolysis by thermolysin or subtilisin in absence or presence of nucleotides.

In the absence of nucleotides two highly stable fragments were recovered (Figure


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5A). Fragments were N-terminally sequenced and identified by mass spectrometry

(Figure 5B). The larger degradation products (3-331; 3-351) corresponded to the N-

terminal domain of ClpB and the core domain of the first AAA module. The second

fragment (536-756) represented the core domain of the second AAA module.

Addition of ATP or ADP slowed the proteolysis considerably and resulted in

stabilisation of fullength ClpB and the occurrence of two other stable cleavage

products. The first fragment (3-331; 3-351) was already obtained by cleavage in the

absence of nucleotide, while the second fragment (537-857; 551-857) corresponded

now to the complete second AAA module. In the presence of ATPγS cleavage was

further limited and reduced amounts of the smaller fragments were obtained (Figure

5A).

Partial proteolysis of ClpB variants revealed that nucleotide-dependent stabilisation of

wild type ClpB can be attributed to hexamer formation. Mutants with oligomerization

deficiencies (K212A, ClpB ∆SSD) or reduced stability of hexamers (ClpB ∆410-532)

were degraded more quickly in the presence of ATP compared to WT ClpB and thus

did not exhibit stabilisation of the fullength version (Figure 5C). The observed defects

of ClpB ∆410-532, missing the “linker” region, in ATP-dependent stabilization could

again be attributed to the presence of the two additional amino acids at the domain

boundary since the control construct (ClpB ∆LBL) with the reinserted linker region

exhibited the same reduced proteolytic stability. Isolated AAA domains (1-409; 551-

857) did not react to ATP addition and were processed rapidly to stable cleavage

products (similar to the fragments obtained for WT ClpB in the absence of

nucleotides). In contrast, a longer version of the first AAA domain (1-567) in

presence of ATP exhibited some protection from degradation. Further structural

analysis of ClpB point mutants surprisingly revealed that conformational changes


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within each AAA domain can occur independently of structural deficiencies in the

other AAA module. Thus, the Walker A mutant K212A of the first AAA domain,

although deficient in oligomerization, still exhibited ATP-dependent conformational

changes in the second AAA domain (stabilization of the second AAA module). On

the other hand structural changes within the second AAA domain were not or only

partially observed in the case of K611A and 813AAA815, ClpB mutants which

nevertheless showed stabilization of the fullength protein (Figure 5C). Thus

oligomerization protects fullength ClpB even in case of mutants that do not bind

nucleotide tightly at the second AAA module (K611A; 813AAA815). We conclude

that stabilization of fullength ClpB against proteolytic degradation primarily reflects

binding of ATP to the first AAA domain. These data also demonstrate that the ability

to form oligomers is not per se a sufficient criteria for probing the structural integrity

of ClpB mutants. Structural integrity with respect to oligomerization and stability

during partial proteolysis was only completely preserved in Walker B mutants of

ClpB, while Walker A and the sensor 2 mutants exhibited significant structural

deficiencies. Interestingly, the double Walker B mutant (E279A/E678A) was much

more resistent towards proteolysis than the single Walker B mutants and ClpB wild

type. Since this mutant is deficient in ATP hydrolysis (see below), the occurrence of

the other fragments (3-331; 3-351and 537-857; 551-857) in ClpB wild type (and

various ClpB mutants) can be attributed to ATP-hydrolysis occurring during the

digestion reaction. These data also indicate that ClpB therefore adopts a different

conformation if both AAA domains have ATP bound, as compared to the situation

where only one AAA domain has ATP bound, while the other domain has ADP

bound.


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ATPase-activities of ClpB mutants and deletion variants

ATPase-activities of ClpB variants were determined in the absence or presence of the

artificial substrate casein, which is known to stimulate ATP hydrolysis by ClpB (Woo

et al., 1992). Wild type ClpB exhibited an ATPase-rate of 0.021/sec and the ATPase-

activity was increased by three to four fold in presence of saturating concentrations of

α-casein (20-fold excess over ClpB monomers). ClpB ∆N had a slightly increased

ATPase activity but was less stimulatable by casein, in agreement with published data

(23,26). Removal of the “linker” region (ClpB ∆410-532) strongly decreased the basal

ATPase-activity by 4-fold, while stimulation by casein was not affected. Similar

results were obtained with the control construct ClpB ∆LBL, bearing a reinserted

linker, indicating that the reduced basal ATPase-activity is primarily caused by the

observed oligomerization deficiencies. All other deletion variants (1-409; 1-567; 551-

857; 1-758) with even stronger defects in oligomerization did not exhibit any ATPase-

activity, even in presence of casein, indicating that ATP-binding and/or hydrolysis is

strictly linked to the formation of hexamers. Consistent with this hypothesis, the basal

ATPase-activities of mutants with oligomerization defects were sensitive to the buffer

conditions: ATP-activities of K212A and R332 were enhanced 2 –3-fold in low salt

buffer, conditions which favour oligomerization (data not shown). In agreement with

these findings addition of 100 mM (NH4)2S04 or 300 mM NH4Cl diminished the

ATPase-rate of fullength ClpB or ClpB ∆N three and five fold, respectively (Table 2,

data not shown). Further analysis of Walker A and Walker B single mutants, bearing

only one functional AAA domain, revealed that especially the first AAA domain was

sensitive towards high ionic strength conditions. Addition of 100 mM (NH4)2S04

strongly reduced the ATPase-activity of mutants, in which the second AAA domain


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was inactivated (K611A; E678A), whereas variants with only an active second AAA

module (E279A; K211A) were only partially affected (Table 2), thereby underlining

the functional importance of the first AAA domain for ClpB assembly. Higher

concentrations of ammonium sulfate (200 mM) completely inhibited ATP-hydrolysis

by ClpB (data not shown).

Single Walker A or Walker B mutations resulted in a complete loss of ATPase-

activity in the corresponding AAA domain, since double Walker A or Walker B

mutants were deficient in ATP-hydrolysis, even in presence of casein. Since

significant ATPase-activities were measurable for all Walker A and Walker B single

mutants, both ATPase-domains seem to contribute to the basal ATPase-activity of

ClpB. Additionally both AAA modules were stimulated to similar degrees by casein.

Variations in the basal ATPase-activities of ClpB point mutants compared to wild

type ClpB indicate a communication between both AAA domains. The basal ATPase-

rate of ClpB E678A was 4-fold increased compared to wild type. However, analysis

of other ClpB mutants revealed that no easy conclusions with respect to the

directionality of the communication between the AAA domains can be drawn.

Mutating the “Arg finger” of the second AAA module (R756A) resulted in a nearly

complete loss of ATPase-activity, although the overall structural integrity was

preserved as for the single Walker B mutant (E678A). The signalling between both

AAA domains seems to be rather complex and subtle conformational changes within

ClpB mutants can obviously have completely different consequences on the other

AAA module.

The mechanism of ATPase-stimulation by casein is unknown. Since ATP-hydrolysis

by ClpB is sensitive to its oligomeric status we tested the possibility that interaction of

ClpB with casein stabilises the hexameric state, thereby increasing the ATPase-


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activity. We compared the ATPase-activity of ClpB concentration at different protein

concentrations both in the absence and presence of casein. In the absence of substrate,

low ClpB-concentrations (0.1-0.2 µM) had no or a strongly reduced specific ATPase-

activity (Figure 6). Increasing ClpB-concentrations activated ATP hydrolysis in a

cooperative manner as revealed by the sigmoidal shape of the curve. In contrast, in the

presence of casein high ATPase-activities were already determined at low ClpB

concentrations (0.1 µM), implying that stabilization of ClpB hexamers by substrates

causes induction of the ATPase activity. Such stabilization by casein was indeed

observed by gelfiltration analysis for the ClpB Walker B mutant E279A/E678A,

which is deficient in ATP-hydrolysis. (Schlieker C., Weibezahn, J., Bukau B. and

Mogk A.; manuscript in preparation). Besides stabilization of the oligomeric state,

conformational changes within ClpB, triggered by substrate binding, may also

contribute to the stimulation of ATP-hydrolysis, since saturating ClpB-concentrations

(1 µM) still showed a lower specific ATPase-activity in the absence of casein (Figure

6).

Chaperone activities of ClpB derivatives

We tested the chaperone activities of the various ClpB variants in vitro by following

the ClpB/DnaK dependent reactivation of heat-aggregated malate dehydrogenase

(MDH) and firefly luciferase. Resolubilization of MDH aggregates by ClpB/DnaK

was directly followed by measuring the decrease of aggregate turbidity in light

scattering experiments. Disaggregation of aggregated luciferase was followed by

determining the refolding rate of luciferase in the presence of the bi-chaperone

system. All disaggregation reactions were performed in the presence of non-saturating


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ClpB concentrations which allowed for sensitive detection of potential activity

defects. Results are summarized in Table 3.

All ClpB deletion variants (1-409; 1-567; 551-857; 1-758) with severe

oligomerization defects were inactive in MDH and luciferase disaggregation. Deletion

of the “linker” region (ClpB ∆410-532) also caused a complete loss of the

disaggregation activity of ClpB. Importantly this inactivation was not caused by the

observed oligomerization deficiency of ClpB ∆410-532, since the control construct

ClpB ∆LBL with the reinserted linker region exhibited significant disaggregation

activity (60 % of ClpB wild type; data not shown). ClpB ∆N had the same chaperone

activity as fulllength ClpB. Full disaggregation activity of ClpB ∆N was also

observed when the ClpB concentrations in the activity assays were further reduced,

thereby increasing the dependency of the disaggregation on ClpB (data not shown).

ClpB variants with point mutations in conserved motifs exhibited no or only partial

chaperone activity (Table 3). In general a disaggregation activity was only observed

for mutants which are still able to form oligomers. Thus the Walker A (K212A) and

Arg finger (R332A) mutations of the first AAA domain, which are deficient in

hexamer formation, were inactive. In contrast, the corresponding mutations in the

second AAA domain (K611A; R756A) exhibited low chaperone activities to varying

degrees (below 10% of the WT control). Partial activities were also obtained in case

of single Walker B mutants, while the double mutant (E279A/E678A) did not have

any disaggregation activity. These findings indicate that while ATP-hydrolysis is

strictly required for chaperone activity, a lack of ATP hydrolysis in one AAA domain

does not completely inhibit the disaggregation activity.

Disaggregation activities of all ClpB variants were also tested in vivo by

complemention of the phenotypes of ∆clpB mutant cells by IPTG-induction of


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plasmid-encoded clpB mutant alleles. Resolubilization of protein aggregates, formed

during severe heat-stress to 45°C in E. coli cells, is severely affected in ∆clpB

mutants. This deficiency is directly linked to a strongly reduced survival rate of ∆clpB

mutant cells at lethal (50°C) temperatures compared to wild type cells

(thermotolerance). We therefore tested the ClpB variants for their ability to re-

establish protein disaggregation and thermotolerance in ∆clpB mutant cells (Figure 7).

Complementation studies were performed in presence of 25 µM IPTG, leading to

three to four fold increased ClpB levels as compared to heat-shocked wild type cells

lacking any plasmid (data not shown). In summary the in vivo disaggregation

activities reflected those observed in vitro: Only wild type ClpB and the ClpB ∆N

could efficiently mediate the resolubilization of protein aggregates and the

development of thermotolerance (Figure 7, Table 3). Constructs with partial

chaperone activities in vitro, such as E279A, also exhibited some activity in vivo.

Discussion

Here we present a detailed structure-function analysis of the AAA+ chaperone ClpB,

which mediates resolubilization of protein aggregates in cooperation with the DnaK

chaperone system. Oligomerization of ClpB was dependent on both AAA domains,

however, each domain seemed to play a different role in ClpB assembly. Deletion of

the α-helical C-domain of the second AAA domain (ClpB ∆SSD) resulted in a severe

defect in oligomerization defect. ClpB ∆SSD stayed predominantly monomeric even

in cross-linking experiments. On the other hand we could demonstrate that ATP

binding to the first AAA domain was also necessary for stabilising the ClpB hexamer.

Several observations support this model. Firstly, the Walker A (K212A) and Arg

finger (R332A) mutants of the first AAA module did not form stable hexamers in the


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presence of ATP, whereas corresponding mutations in the second NBD (K611A,

R756A) had no influence on ClpB oligomerization, although these mutants exhibited

severe structural deficiencies. Secondly, the introduction of amino acids into the C-

domain of the first AAA module (ClpB ∆410-532; ClpB ∆LBL) also resulted in

destabilization of ClpB oligomers, underlining the importance of this domain in

hexamerization. We also could show that high salt conditions (addition of 100mM

(NH4)2S04) causes dissociation of ClpB oligomers by interfering predominantly with

subunit contacts between the first AAA domains. Interestingly, the recently

determined structure of monomeric ClpA suggests, that electrostatic interactions

between the first AAA domains of ClpA could play a much more important role than

the second AAA modules in protein oligomerization (28). These findings might also

explain the observed salt-sensitivity of ClpB oligomerization and underline the

functional importance of the first AAA domain in this process. Finally, the observed

changes in fluorescence of W543, located in the C-domain of the first AAA module,

revealed a conformational rearrangement of this region in response to nucleotides.

The observed blue shift (4 nm) in the wavelength for maximum fluorescence could be

attributed to ClpB oligomerization. We suggest that changes in W543 fluorescence

reflect interactions of the first C-domain not only with itself but also an adjacent

ATPase-domains, thereby leading to increased shielding of W543 and stabilization of

the oligomer. Nucleotide-dependent conformational changes of the C-terminal α-

helical domain was also demonstrated by limited proteolysis. While the C-domain

was rapidly degraded in the absence of nucleotides, it became largely resistent to

proteases upon nucleotide addition (Figure 5B). Similarly the C-domain of the second

AAA module was also protected by addition of nucleotides. This protection is likely

to be caused by the interaction of C-domains with their own AAA domain and that of


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adjacent subunits, thereby becoming less accessible to proteases. Consistently, ClpB

mutants with defects in nucleotide binding (K212A, K611A, 813AAA815) did not

exhibit stabilization of the corresponding C-domains.

A functional importance of the first ATPase domain for Hsp100 oligomerization has

also been reported for E. coli ClpA (29,30) and ClpB from Thermus thermophilus

(31). It is intriguing that the contributions of the individual AAA domains differ in the

ClpB homologues Hsp104 and Hsp78 from S. cerevisiae. Here mutations of the Lys

residue in the Walker A motif of the second AAA domain resulted in a loss of ability

to form hexamers (32-34).

Hexamerization of ClpB is a prerequisite for its chaperone activity. Since ATP is

bound at the interface of two neighbouring subunits in the hexamer, oligomerization

additionally influences the ATPase activity of ClpB. Consequently high salt

conditions inhibit ATP-hydrolysis by ClpB. Furthermore ClpB variants with defects

in oligomerization exhibited little or no ATPase-activity. On the other hand, the

stimulation of ATP-hydrolysis by substrates, such as α-casein, is most likely due to

the stabilization of ClpB oligomers.

Mutations of conserved Lys and Glu residues in the Walker A and Walker B motifs of

both AAA modules completely abolished ATP hydrolysis by the corresponding AAA

domain. Since single mutants still retained significant ATPase-activity, both AAA

domains seem to contribute to the basal rate of ATP hydrolysis by ClpB.

Interestingly, ClpB homologues from different organisms differ significantly in their

ATPase-cycle. While both AAA domains of ClpB from T. thermophilus (31) also

contribute to the basal ATPase-activity, ATP-hydrolysis by the yeast homologue

Hsp104 is dominated by the first AAA module (25). Such differences may potentially


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explain the observed species specificity in the cooperation of ClpB/Hsp104 proteins

with the corresponding Hsp70 partners (35). Variations in the basal ATPase-activity

of ClpB mutants indicate communication between both AAA domains. However,

different mutations in the same AAA domain (E678A; R756A) exhibited different

influences on the ATPase-activity of the other AAA module. The signalling between

both AAA domains appears to be rather complex and subtle conformational changes

within ClpB mutants can have completely different consequences on the other AAA

module.

The function of the N-domain still remains enigmatic. Isolated N-domains from ClpA

and ClpB have been shown to form stable, monomeric domains, which are probably

separated from the AAA domains by a flexible linker (7,36,37). Consistent with this

model, N-domains are not essential for oligomerization of ClpA or ClpB (23,36) (this

work). The connector is apparently sterically inaccessible to proteases since partial

proteolysis of ClpB did not release significant amounts of isolated N-domains, but

rather produced a fragment comprising the N-domain and the core domain of the first

AAA module (1-353). Similar findings have been reported for ClpA (36). The

reported consequences of N-terminal deletions of ClpB on its chaperone activity are

contradictory. Zolkiewski and co-worker showed that a ClpB variant, starting from

the internal start site (149-857), was completely inactive in refolding of aggregated

luciferase (23). In contrast, a ClpB ∆N (143-857) variant showed the same chaperone

activity as wild type ClpB both in vitro and in vivo (resolubilization of protein

aggregates, development of thermotolerance). In agreement with these data, an N-

terminal truncated ClpB derivative of Synechococcus sp. PCC7942 conferred the

same degree of thermotolerance in vivo as fullength ClpB and likewise the N-terminal


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truncated version of Thermus thermophilus ClpB was also shown to be active in

protein disaggregation in vitro (38,39). The existence of ClpB homologues completely

lacking an N-domain in Mycoplasma sp. also argues against an essential function of

the N-domains in the disaggregating activities of ClpB (40,41).

What might be the function of the N-domain with respect to these conflicting results?

N-domains have been proposed to mediate substrate binding, however the reported

activities of N-terminally truncated ClpB variants clearly rule out an essential

function in this process. Interestingly, defects in substrate binding were also not the

basis of the inactivation of ClpB 149-857, since this deletion variant interacted with

casein and unfolded luciferase indistinguishable from ClpB wild type (37). N-

domains may be therefore involved in a mechanism for coupling the ATPase-cycle of

ClpB with its proposed unfolding activity. Since a longer deletion of the N-domain,

including parts of the flexible linker to the first AAA domain, has been reported to be

inactive, this linker could potentially contribute to induced structural changes in

bound substrates. Alternatively N-domains may be involved in other ClpB activities,

which are so far unknown and are not related to protein disaggregation. Such new

activities have been described for a second ClpB homologue in Synechococcus, which

is essential for cell viability, but not involved in thermorolerance (42). Interestingly,

the N-domain of this ClpB variant seems to be crucial for its unknown activity and

may serve as binding sites for special substrates or, alternatively, for specific adaptor

proteins. Binding of adaptor proteins to N-domains has been demonstrated for ClpA

(43) and the AAA protein p97 (44,45).

The “linker” region of ClpB was originally suggested to separate both AAA domains

(1). We propose that the “linker” instead interrupts the C-domain of the first AAA

domain as was recently suggested for the yeast homologue Hsp104 (46). A


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comparison of different ClpB fragments (1-409; 1-567) with respect to their

oligomerization and their resistence to proteolysis supports this model. Firstly, cross-

linking studies revealed that ClpB 1-567 can form dimeric species in contrast to the

shorter variant (1-409), which remains monomeric under all conditions. Additionally

partial proteolysis revealed a more pronounced protection of the fullength version (1-

567), which was not observed for ClpB 1-409. Formation of dimeric species and the

observed partial stabilization is likely to be caused by the interaction of the helical C-

domain, which can only be formed in ClpB 1-567, with its own and an adjacent AAA

domain. Interestingly a very similar domain organization has been proposed for ClpA

(36). In ClpA, which is missing the “linker” region, a short basic loop (KRKK) is also

inserted into the C-domain of the first AAA module.

The “linker” region of ClpB, in contrast to the N-domain, is essential for chaperone

activity. It is proposed to form a 4-times repeated coiled-coil (47) which is very likely

to play an important role in ClpB function. Conformational changes in response to

nucleotides within the linker region were shown by its increased proteolytic stability

in the context of fullength ClpB. We assume that the “linker” is not an integral part of

the ClpB hexamer, since oligomerization was still possible in case of a ClpB variant

missing the “linker” region (ClpB ∆410-532). Similarly, the I-domain of HslU,

although inserted into the AAA domain, forms an independet structural domain and is

exposed at the surface of the oligomer (3,4). The function of the linker region is still

unknown. The postulated coiled-coil structure might be involved in protein-protein

interaction. Since the ATPase-activity of ClpB ∆410-532 was still strongly

stimulatable by casein, the linker region cannot serve as a primary substrate binding

site, at least for this type of substrate. Alternatively, the “linker” region is necessary


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for coupling ATP hydrolysis and substrate unfolding, as proposed recently by

Lindquist and co-workers (46).


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References

1. Schirmer, E. C., Glover, J. R., Singer, M. A., and Lindquist, S. (1996) Trends

Biochem Sci 21(8), 289-96.

2. Neuwald, A. F., Aravind, L., Spouge, J. L., and Koonin, E. V. (1999) Genome

Res 9(1), 27-43

3. Bochtler, M., Hartmann, C., Song, H. K., Bourenkov, G. P., Bartunik, H. D.,

and Huber, R. (2000) Nature 403(6771), 800-5.

4. Sousa, M. C., Trame, C. B., Tsuruta, H., Wilbanks, S. M., Reddy, V. S., and

McKay, D. B. (2000) Cell 103(4), 633-43.

5. Li, J., and Sha, B. (2002) J Mol Biol 318(4), 1127-37.

6. Smith, C. K., Baker, T. A., and Sauer, R. T. (1999) Proc. Natl. Acad. Sci. USA

96, 6678-6682

7. Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci 10(3), 551-9.

8. Banecki, B., Wawrzynow, A., Puzewicz, J., Georgopoulos, C., and Zylicz, M.

(2001) J Biol Chem 276(22), 18843-8.

9. Kim, Y. I., Levchenko, I., Fraczkowska, K., Woodruff, R. V., Sauer, R. T.,

and Baker, T. A. (2001) Nat Struct Biol 8(3), 230-3.

10. Goloubinoff, P., Mogk, A., Peres Ben Zvi, A., Tomoyasu, T., and Bukau, B.

(1999) Proc. Natl. Acad. Sci. USA 96, 13732-13737

11. Mogk, A., Tomoyasu, T., Goloubinoff, P., Rüdiger, S., Röder, D., Langen, H.,

and Bukau, B. (1999) EMBO J. 18, 6934-6949

12. Motohashi, K., Watanabe, Y., Yohda, M., and Yoshida, M. (1999) Proc. Natl.

Acad. Sci. USA 96, 7184-7189

13. Zolkiewski, M. (1999) J. Biol. Chem. 274, 28083-28086


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

14. Laufen, T., Mayer, M. P., Beisel, C., Klostermeier, D., Reinstein, J., and

Bukau, B. (1999) Proc. Natl. Acad. Sci. USA 96, 5452-5457

15. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal Chem 68(5),

850-8.

16. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis,

T., and Mann, M. (1996) Nature 379(6564), 466-9.

17. Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P., and Bukau, B. (2001)

Mol Microbiol 40(2), 397-413.

18. Kim, K. I., Cheong, G. W., Park, S. C., Ha, J. S., Woo, K. M., Choi, S. J., and

Chung, C. H. (2000) J Mol Biol 303(5), 655-66.

19. Ogura, T., and Wilkinson, A. J. (2001) Genes Cells 6(7), 575-97.

20. Nicholls, A., Sharp, K. A., and Honig, B. J. (1991) Proteins 11, 281-296

21. Karata, K., Inagawa, T., Wilkinson, A. J., Tatsuta, T., and Ogura, T. (1999) J.

Biol. Chem. 274, 26225-26232

22. Zolkiewski, M., Kessel, M., Ginsburg, A., and Maurizi, M. R. (1999) Protein

Sci 8(9), 1899-903.

23. Barnett, M. E., Zolkiewska, A., and Zolkiewski, M. (2000) J Biol Chem

275(48), 37565-71.

24. Schmid, F. X. (1991) Curr. Biol. 1, 36-41

25. Hattendorf, D. A., and Lindquist, S. L. (2002) Embo J 21(1-2), 12-21.

26. Park, J. H., Lee, Y. S., Chung, C. H., and Goldberg, A. L. (1988) J. Bacteriol.

170, 921-926

27. Konieczny, I., and Liberek, K. (2002) J Biol Chem 277(21), 18483-8.

28. Guo, F., Maurizi, M. R., Esser, L., and Xia, D. (2002) J Biol Chem 277(48),

46743-52.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29. Singh, S. K., and Maurizi, M. R. (1994) J Biol Chem 269(47), 29537-45.

30. Seol, J. H., Baek, S. H., Kang, M.-S., Ha, D. B., and Chung, C. H. (1995) J.

Biol. Chem. 270, 8087-8092

31. Watanabe, Y. H., Motohashi, K., and Yoshida, M. (2002) J Biol Chem 277(8),

5804-9.

32. Parsell, D. A., Kowal, A. S., and Lindquist, S. (1994) J Biol Chem 269(6),

4480-7.

33. Schirmer, E. C., Queitsch, C., Kowal, A. S., Parsell, D. A., and Lindquist, S.

(1998) J. Biol. Chem. 273, 15546-15552

34. Krzewska, J., Konopa, G., and Liberek, K. (2001) J Mol Biol 314(4), 901-10.

35. Krzewska, J., Langer, T., and Liberek, K. (2001) FEBS Lett 489(1), 92-6.

36. Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and

Maurizi, M. R. (2001) J Biol Chem 276(31), 29420-9.

37. Tek, V., and Zolkiewski, M. (2002) Protein Sci 11(5), 1192-8.

38. Clarke, A. K., and Eriksson, M. J. (2000) J Bacteriol 182(24), 7092-6.

39. Beinker, P., Schlee, S., Groemping, Y., Seidel, R., and Reinstein, J. (2002) J

Biol Chem 277(49), 47160-6.

40. Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A.,

Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G., Kelley, J. M.,

and et al. (1995) Science 270(5235), 397-403.

41. Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C., and Herrmann,

R. (1996) Nucleic Acids Res 24(22), 4420-49.

42. Eriksson, M. J., Schelin, J., Miskiewicz, E., and Clarke, A. K. (2001) J

Bacteriol 183(24), 7392-6.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

43. Dougan, D. A., Reid, B. G., Horwich, A. L., and Bukau, B. (2002) Mol Cell

9(3), 673-83.

44. Rouiller, I., Butel, V. M., Latterich, M., Milligan, R. A., and Wilson-Kubalek,

E. M. (2000) Mol Cell 6(6), 1485-90.

45. Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D., and Warren, G. (2000)

Embo J 19(10), 2181-92.

46. Cashikar, A. G., Schirmer, E. C., Hattendorf, D. A., Glover, J. R.,

Ramakrishnan, M. S., Ware, D. M., and Lindquist, S. L. (2002) Mol Cell 9(4),

751-60.

47. Celerin, M., Gilpin, A. A., Schisler, N. J., Ivanov, A. G., Miskiewicz, E., Krol,

M., and Laudenbach, D. E. (1998) J Bacteriol 180(19), 5173-82.


http://ww

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Figure legends

Figure 1

Proposed domain organization of E. coli ClpB and conserved motifs, involved in

ATP-binding and ATP-hydrolysis. ClpB consists of an N-terminal domain, two

nucleotide binding domains (ATP-1; ATP-2), which are separated by a “linker”

region, and a C-terminal SSD. Both nucleotide binding domains contain the Walker A

(206GX4GKT213; 605GX4GKT612) and Walker B (276Hy2DE279; 675Hy2DE678)

motifs where X = any amino and Hy = hydrophobic amino acids. Conserved Arg

residues (R) present in both nucleotide binding domains (R332; R756), are proposed

to serve as “Arg fingers”, contacting the ATP bound to an adjacent subunit. The

invariant sensor 2 motif (813GAR815) of the SSD of Hsp100 proteins potentially

sense the nucleotide status of ATP-2. Conserved residues of these motifs (marked in

bold) were subjected to alanine mutagenesis, as indicated by arrows.

Figure 2

Oligomerization of ClpB wild type and mutant derivatives analyzed by gel filtration

chromatography. A) Elution profiles of ClpB wild type (WT) were recorded in the

absence (-) or presence of nucleotides (2mM ATP/ADP) in the running buffer (50

mM Tris, pH 7.5; 20 mM MgCl2; 150 mM KCl; 10% (v/v) glycerol). Elution

positions of protein standards (thyroglobulin (669 kDa), ferritin (440 kDa) catalase

(232 kDa) and IgG (158 kDa) are given. B) Elution profiles of the indicated ClpB

mutants were recorded in presence of ATP (2 mM) in the running buffer.


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Figure 3

Assembly of ClpB wild type and derivatives revealed by cross-linking. (A) 1 µM

ClpB wild type (WT) and the indicated point mutants were incubated for 5 min at

30°C in the absence of nucleotides (2) or the presence of 2 mM ATP (3) or ADP (4).

Cross-linking reactions were initiated by addition of glutaraldehyde and proceeded for

10 min. Cross-linked proteins were separated on SDS (4-12 %) polyacrylamide gels,

followed by silver staining. Incubation of ClpB proteins in the absence of cross-linker

(1) served as control. (B) ClpB deletion variants were incubated in the absence (2) or

presence of 2 mM ATP (3) for 5 min at 30°C. Cross-linking was performed as

described above. Incubation of truncated ClpB species in the absence of cross-linker

(1) served as control.

Figure 4

(A) Nucleotide-dependent blue shift in the wavelength of maximum ClpB

fluorescence. Emission spectra of tryptophan fluorescence of ClpB (0,5 µM) in the

absence of nucleotide (black line) or the presence of 2 mM ATP (grey line) or ADP

(thin black line) in buffer A (50 mM Tris, pH 7.5; 150 mM KCl; 20 mM MgCl2; 2

mM DTT) are shown. Additionally ClpB fluorescence was recorded in low salt buffer

(buffer A without 150 mM KCl)(black dotted line) or in the presence of 2 mM ATP in

high salt buffer (buffer A plus 100 mM (NH4)2S04) (grey dotted line). The maximums

in the wavelength of tryptophan fluorescence are indicated. (B) High salt

concentrations inhibit cross-linking of ClpB to oligomeric species. Cross-linking of

ClpB (1 µM) by glutaraldehyde was performed in the absence or presence of ATP (2

mM) and/or (NH4)2S04 (100 mM). Cross-linked proteins were separated on SDS (4-12

%) polyacrylamide gels, followed by silver staining.


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Figure 5

Nucleotide-dependent structural changes in ClpB revealed by limited proteolysis. (A)

Partial proteolysis of ClpB wild type by thermolysin and subtilisin was performed in

the absence of nucleotide or in the presence of 2 mM ATP/ADP/ATPγS for 60 min.

Cleavage products were separated on SDS (15 %) polyacrylamide gels, followed by

silver stainin. Cleavage sites were identified by N-terminal sequencing and mass

spectrometry. (B) Proposed domain organization of ClpB. Domains were defined

from sequence analysis, secondary structure prediction and results obtained from

partial proteolysis. ClpB domains are: N-domain (1-144), ATP-1 (144-340), C-1

(341-409 and 525-550), the “linker” region (410-525), ATP-2 (550-755) and C-2

(755-857). ATP-1/2 correspond to the core domains of the AAA modules. C-1/2

represent the C-terminal helical domains of the AAA modules. Sites of cleavage by

thermolysin and subtilisin are indicated (open arrows: cleavage site in absence of

ATP; filled arrows: cleavage sites in presence of nucleotides). (C) Partial proteolysis

of ClpB mutants and fragments by thermolysin. Proteins were incubated in the

absence or presence of ATP for 60 min. Cleavage products were separated on SDS

(15 %) polyacrylamide gels, followed by silver staining.

Figure 6

The ATPase-activity of ClpB depends on protein concentration and protein substrates.

The specific ATPase-activity of ClpB was measured at different protein

concentrations in the absence or presence of α-casein (0.25 mg/ml).


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Figure 7

In vivo activity of ClpB derivatives. (A) E. coli wild type (MC4100), ∆clpB mutant

cells and ∆clpB mutants, bearing plasmid-encoded clpB alleles under the control of an

IPTG-regulatable promotor, were grown in LB medium at 30°C in the presence of 25

µM IPTG to mid-exponential phase. Afterwards strains were heat-shocked to 50°C

and incubated for the indicated time. Various dilutions of the stressed cells were

spotted on LB plates and incubated at 30°C. After 24 h, colony numbers were

counted, and survival rates were calculated in relation to unstressed cells. (B) Cultures

of ∆clpB mutant strains, bearing plasmid-encoded clpB alleles under the control of an

IPTG-regulatable promotor, were grown in LB medium in the presence of 25 µM

IPTG at 30°C to logarithmic phase. Cells were then shifted to 45°C for 30 min,

followed by a recovery phase at 30°C for 60 min. Aggregated proteins were isolated

before (0 min) and after the recovery phase (60 min) and analyzed by SDS-PAGE

followed by staining with Coomassie Brilliant Blue.


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Table 1: Oligomerization of ClpB fragments and mutants

ClpB fragments

gel filtration

chromatography

Cross-linking

products

Maximum of ClpB

fluorescence

1–857 (wild type) hexamer hexamers 346.5

143-857 (∆N) hexamer hexamers 346.5

1-758 (∆SSD) monomer monomers-

trimers

351

∆410-532 (∆linker) hexamer hexamers 348.5

1-409 monomer Monomer n.d.

1-567 monomer Monomer n.d.

551-857 monomer Monomers-

dimers

n.d.

ClpB mutants

K212A (Walker A) monomer monomers-

tetramers

351

K611A (Walker A) hexamer hexamers 347

K212A/K611A dimer monomers-

tetramers

351

E279A (Walker B) hexamer hexamers 346.5

E678A (Walker B) hexamer hexamers 346.5

E279A/E678A hexamer hexamers 346.5

R332A (Arg finger) dimer monomer-

hexamers

348.5

R756A (Arg finger) hexamer hexamers 346.5

813AAA815 (Sensor 2) hexamer hexamers 346

Assembly of the indicated ClpB derivatives was analyzed in the presence of 2 mMATP by gel filtration chromatography, cross-linking and tryptophan fluorescence.Size of ClpB oligomers were calculated from a calibration curve with proteinstandards. Cross-link products formed after 10 min incubation with glutaraldehyde,were analysed by SDS 4-12% polyacrylamide gel electrophoresis. Most populatedspecies are underlined. ATP-dependent oligomerization of ClpB is coupled to a blue-shift in the maximum of fluorescence (346.5 nm instead of 350.5 nm in the absence ofnucleotide). The maximum of ClpB fluorescence is indicated.


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Table 2: ATPase-activities of ClpB fragments and mutants

ATPase-activities of the indicated ClpB derivatives were determined in buffer A with

or without 150 mM KCl (high and low salt, respectively). The factors of ATPase-

stimulation in the presence of 0.25 mg/ml α-casein and the factors of ATPase-

inhibition in the presence of 100 mM ammonium sulfate are given.

ClpB fragments

ATPaseactivity

(1/s)

Factor of ATPase-

stimulation by

αααα -casein

Factor of ATPase-

inhibition by

sulfate

1–857 (wild type) 3.3 3

143-857 (∆N) 1.9 4.9

1-758 (∆SSD) 0 n.d.

∆410-532 (∆linker) 6 n.d.

1-409 0 n.d.

1-567 0 n.d.

551-857 0 n.d.

ClpB mutants

0.021

0.037

0

0.005

0

0

0

K212A (Walker A) 4.4 1.4

K611A (Walker A) 5.3 4.2

K212A/K611A 0 n.d.

E279A (Walker B) 3.8 1.7

E678A (Walker B) 2.7 5.3

E279A/E678A 0 n.d.

R332A (Arg finger) 2.4 n.d

R756A (Arg finger) 0 n.d

813AAA815 (SSD motif)

0.006

0.019

0

0.023

0.089

0

0.019

0.003

0.036 2.8 n.d

n.d.: not determined


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Table 3: Chaperone activities of ClpB mutants and fragments

Chaperone activity (disaggregation rate of aggregated MDH; refolding rate of

aggregated Luciferase) of ClpB wild type was set as 100%. In vivo activity reflects

the ability of ClpB derivatives to restore thermotolerance and protein disaggregation

in E. coli ∆clpB mutant cells (+, full activity; (-) residual activity; -, no activity)

ClpB fragments

Disaggregation of

aggregated MDH

(% activity)

Refolding of

aggregated Luciferase

(% activity)

in vivo

activity

1–857 (wild type) 100 100 +

143-857 (∆N) 98 99 +

1-758 (∆SSD) 0 0 -

∆410-532 (∆linker) 0 0 -

1-409 0 0 n.d.

1-567 0 0 n.d.

551-857 0 0 n.d.

ClpB mutants

K212A (Walker A) 0 0 -

K611A (Walker A) 4-5 8-10 (-)

K212A/K611A 0 0 -

E279A (Walker B) 7-9 8-10 (-)

E678A (Walker B) 2-3 4-5 (-)

E279A/E678A 0 0 -

R332A (Arg finger) 0 0 -

R756A (Arg finger) 0.5-1 0.5-1 -

813AAA815 (SSD motif) 0 0 -

n.d.: not determined


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1 149 409 530 758 857N ATP-1 Linker ATP-2

aa

ClpB

GAR

AAA

SSD

A B

GX4GKT Hy2DE

A A

A B

GX4GKT Hy2DE

A A

R

A

R

A

Figure 1, Mogk et al

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10 11 12 1390

0,04

0,08

0,12

elution volume [ml]

AU

(29

0 nm

)

protein standard [kDa]669 440 232 158

K611A

K212A

K212A/K611A

E279A

E678A

E279A/E678A

WT


ATP ADP -

A

B

10 11 12 139

elution volume [ml]


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∆N ∆SSD

1 2 3 1 2 3 1 2 3 1 2 3

∆Linker

1-409 1-567 551-857143-857 1-758∆410-532

1 2 31 2 3

A

B

WT K212A K611A K212/K611A E279A E678A E279A/

E678A

1 2 3 41 2 3 4 1 2 3 41 2 3 41 2 3 41 2 3 41 2 3 4


Walker A Walker B

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170

175

180

185

190

195

200

205

335 340 345 350 355 360 365

rela

tive

inte

nsit

ies

nm

no nucleotide

ATP

ADP

low salt

ATP + high salt

346,5 350,5A

BATP

(NH4)2SO4X-Linker

---

--+

+-+

-++

+++

Monomer

Hexamer



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Thermolysin

Subtilisin

N ATP-1 C-1 ATP-2 C-2Linker

1 144

340 760550

410 530

A

B

C

9766

39

26

21

14

ClpB

3-331

536-756591-756

551-857

ClpB

536-756

3-351

551-857537-857

N-domain

- ATP ADPATPγS

Thermolysin Subtilisin

- ATP

- ATP

+ ATP

WT

Walker A Walker B14

3-85

7

1-75

8

1-40

9

∆410

-532

551-

857

1-56

7

K21

2A

K61

1A

E27

9A

K21

2A/

K61

1A

E67

8AE

279A

/E

678A

R33

2A

R75

6A

813A

AA

815

ClpB

3-331

536-756591-756

551-857

3-331

536-756

591-756


no nucleotide ATP/ADP


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0

0.02

0.04

0.06

0.08

0 0.25 0.5 0.75 1

AT

Pas

e-ac

tivi

ty (

1/s)

ClpB-conc. [µM]

+ α-casein

− α-casein



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% v

iabl

e ce

lls

time at 50°C (min)

0.1

1

10

100

0 30 60 90 120


A

B

0 60 0 60 0 60 0 60 0 60ClpB ClpB ∆N E279A E678A

E279A/E678A

97

66

39

26

time (min)

E. coli MC4100∆clpBClpB (1-857)ClpB ∆N (143-857)ClpB E279AClpB E678AClpB E279A/E678A


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0

1

2

3

4

5

6

7

8

9

0 5 10 15 20 25 30 35 40

α-caseinClpB6/α-casein

α-caseinα-casein +ClpB E279A/E678A

0

2

4

6

8

10

12

14

0 5 10 15 20 25 30 35 40

ClpBClpB6

ClpB E279A/E678A

ClpB E279A/E678A+ α-casein

A

B

Supplementary figure

% 3

H-c

asei

n

fractions

fractions

% 3

H-C

lpB

E27

9A/E

678A


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Bernd BukauAxel Mogk, Christian Schlieker, Christine Strub, Wolfgang Rist, Jimena Weibezahn and

oligomerization, ATP-hydrolysis and chaperone activityRoles of individual domains and conserved motifs of the AAA+ protein ClpB in

published online March 6, 2003J. Biol. Chem.

10.1074/jbc.M209686200Access the most updated version of this article at doi:

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Roles of individual domains and conserved motifs of the ... · 2003-03-06 · Axel Mogk 1,2,3, Christian Schlieker 1,2, Christine Strub 1,2, Wolfgang Rist 1,2, Jimena Weibezahn 1,2