From the Zentrum für Molekulare Biologie
Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany and
§ Institut für Biochemie und Molekularbiologie,
Universität Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany
Received for publication, September 20, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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ClpB of Escherichia coli is an
ATP-dependent ring-forming chaperone that mediates the
resolubilization 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
that 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
stabilized the ClpB oligomer. Analysis of mutants of conserved residues
in Walker A and 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 dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the
presence of the linker region, although not involved in
oligomerization, is essential for ClpB chaperone activity.
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), whereas class II proteins
(ClpX and HslU, as representatives of E. coli) contain only
a single NBD (homologous to ATP-2) (1). Sequence analysis of the
NBDs 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, that showed significant
similarity to the AAA proteins N-ethylmaleimide-sensitive
fusion protein 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 that forms the nucleotide binding pocket, containing the classical Walker A and B motifs, and a C-terminal 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 zinc 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 subfamily is the presence/absence of a region that 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 (~140 residues) but is absent in ClpA (see Fig. 1) (1).
ClpB is unique among the Hsp100/Clp proteins because 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 that 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.
Strains and Plasmids--
E. coli strains used were
derivatives of MC4100 (araD139 Proteins--
Wild type and mutant ClpB were purified as
described after overproduction in
Tryptophan Fluorescence--
Measurements of the intrinsic
tryptophan fluorescence of ClpB were performed on a PerkinElmer Life
Sciences 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 [ 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 high pressure liquid
chromatography system (PerkinElmer Life Sciences) connected to a SEC
400 column (Bio-Rad). 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 another 10 min. Reactions were stopped
by addition of 1 M Tris, pH 7.5, and cross-linking products
were analyzed 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, and ATP 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 (PerkinElmer Life Sciences
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 °C for the indicated time. Serial dilutions (10 Design of ClpB Mutants and Deletions Variants--
ClpB is
proposed to consist of an N-terminal domain, two AAA-domains (ATP-1 and
ATP-2), which are separated by a linker region, and the C-terminal SSD
domain (as illustrated in Fig. 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 full-length protein (aa 1-857) and an N-terminally
truncated variant (aa 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 full-length 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 (Val-149). 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 Met-143 as the start site
for ClpB
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 (Lys-212 and Lys-611 of ClpB) contact
the phosphate groups of the
The formation of hexameric rings in AAA+ proteins brings residues of
adjacent protomers into close proximity to ATP, bound to a neighboring
subunit. Such interactions could provide the 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 (Arg-332 and Arg-756 in ClpB)
can potentially act as "Arg fingers" by contacting the ATP bound to
a neighbored subunit (21). All described motifs, involved in ATP
binding and hydrolysis, were subjected to alanine mutagenesis, as
illustrated in Fig. 1. ClpB mutants and deletion variants were purified
and characterized with respect to oligomerization, ATP hydrolysis, and
chaperone activity.
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 (Fig. 2A). In
the absence of nucleotide, ClpB eluted predominantly as a dimer,
although ADP addition led to the formation of trimers and/or tetramers.
Analysis of ClpB deletion variants revealed that isolated AAA-domains
(1-409, 1-567, and 551-857) cannot oligomerize. In agreement with
previous data (23), hexamerization is strictly dependent on the
presence of the SSD domain, although the N-domain and the linker region
are dispensable (Table I).
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,
although corresponding mutations in the second AAA module (K611A and
R756A) domain had no effect (Fig. 2B and Table I). Finally,
all Walker B mutants in the two AAA modules (E279A, E678A, and
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 was obtained within 10 min in the absence of
nucleotide, indicating that ClpB assembly can in principle also occur
without nucleotide. Because such nucleotide-independent hexamerization
was not observed during gel filtration runs, the formed oligomers seem
to be unstable in the absence of ATP.
Kinetic analysis of the cross-linking reaction revealed that a ladder
of cross-linking products preceded 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. Whereas 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 the hexameric and pentameric species, respectively
(Fig. 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 gel filtration analysis; however, qualitative
differences with respect to the oligomerization deficiencies were
revealed. Isolated AAA-domains (1-409 and 551-857) stayed as monomers
in presence of glutaraldehyde, whereas in case of the ClpB
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 (Trp-462 and Trp-543). 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 (Fig. 4A). This
change was more pronounced in the presence of ATP compared with ADP.
Several findings suggest that the observed changes in fluorescence are
caused by oligomerization of ClpB. First, the blue shift was also
observed when ClpB was incubated in low salt buffer in the absence of
nucleotide (Fig. 4). Such buffer conditions have been shown for several
ClpB homologues to stabilize the oligomer in the absence of nucleotide
(25, 26). Consistently, the ATP-dependent blue shift was
reverted under high ionic strength conditions (addition of 100 mM (NH4)2SO4 or 300 mM NH4Cl), conditions which have been shown to
inhibit ClpB activity (10). De-oligomerization of ClpB in the
additional presence of 100 mM
(NH4)SO4 or 300 mM
NH4Cl was also observed in cross-linking experiments. Such
high salt conditions resulted in a strong reduction of ClpB
cross-linking efficiency in the absence or presence of ATP (Fig.
4B and data not shown).
Second, the observed changes in fluorescence also became
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 was only partially
observed for ClpB variants with oligomerization defects (Table I).
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 (Trp-462) was
constructed. Although 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 Trp-543, 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 we checked whether such
structural rearrangements were preserved in the constructed ClpB
variants. ClpB was subjected to limited proteolysis by thermolysin or
subtilisin in the absence or presence of nucleotides. In the absence of
nucleotides two highly stable fragments were recovered (Fig.
5A). Fragments were N-terminally sequenced and identified by mass spectrometry (Fig. 5B). The larger degradation products (aa 3-331 and 3-351)
corresponded to the N-terminal domain of ClpB and the core domain of
the first AAA module. The second fragment (aa 536-756) represented the
core domain of the second AAA module. Addition of ATP or ADP slowed the
proteolysis considerably and resulted in stabilization of full-length
ClpB and the occurrence of two other stable cleavage products. The
first fragment (aa 3-331 and 3-351) was already obtained by cleavage
in the absence of nucleotide, although the second fragment (aa 537-857
and 551-857) corresponded now to the complete second AAA module. In
the presence of ATP
Partial proteolysis of ClpB variants revealed that
nucleotide-dependent stabilization of wild type ClpB can be
attributed to hexamer formation. Mutants with oligomerization
deficiencies (K212A, ClpB 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 (27). Wild type ClpB exhibited an ATPase rate of 0.021/s, and the ATPase activity was increased by 3-4-fold in the presence of saturating concentrations of
Single Walker A or Walker B mutations resulted in a complete loss of
ATPase activity in the corresponding AAA-domain, because double Walker
A or Walker B mutants were deficient in ATP hydrolysis, even in
presence of casein. Because significant ATPase activities were
measurable for all Walker A and Walker B single mutants, both ATPase
domains seemed 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 with wild type ClpB indicate a communication between both
AAA-domains. The basal ATPase rate of ClpB E678A was increased 4-fold
compared with 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
signaling between both AAA-domains seemed 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. Because ATP
hydrolysis by ClpB is sensitive to its oligomeric status, we tested the
possibility that interaction of ClpB with casein stabilizes the
hexameric state, thereby increasing the ATPase 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 activity or a strongly reduced specific ATPase activity (Fig. 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
gel filtration analysis for the ClpB Walker B mutant E279A/E678A, which
is deficient in ATP
hydrolysis.2 Besides
stabilization of the oligomeric state, conformational changes within
ClpB, triggered by substrate binding, may also contribute to the
stimulation of ATP hydrolysis, because saturating ClpB concentrations
(1 µM) still showed a lower specific ATPase activity in
the absence of casein (Fig. 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 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 ClpB concentrations which allowed for sensitive detection of potential activity defects. Results are summarized in
Table III.
All ClpB deletion variants (aa 1-409, 1-567, 551-857, and 1-758)
with severe oligomerization defects were inactive in MDH and luciferase
disaggregation. Deletion of the linker region (ClpB-(
ClpB variants with point mutations in conserved motifs exhibited none
or only partial chaperone activity (Table III). In general a
disaggregation activity was only observed for mutants that 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 and 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, although the double mutant (E279A/E678A) did not have any
disaggregation activity. These findings indicate that whereas 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 complementation of the phenotypes of
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 A functional importance of the first ATPase domain for Hsp100
oligomerization has also been reported for E. coli ClpA (30, 31) and ClpB from Thermus thermophilus (32). It is
intriguing that the contributions of the individual AAA-domains differ
in the ClpB homologues Hsp104 and Hsp78 from Saccharomyces
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 (33-35).
Hexamerization of ClpB is a prerequisite for its chaperone activity.
Because ATP is bound at the interface of two neighboring 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
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. Because 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.
Although both AAA-domains of ClpB from T. thermophilus (26,
32) 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 explain the observed species specificity in
the cooperation of ClpB/Hsp104 proteins with the corresponding Hsp70
partners (36). Variations in the basal ATPase activity of ClpB mutants
indicate communication between both AAA-domains. However, different
mutations in the same AAA-domain (E678A and R756A) exhibited different
influences on the ATPase activity of the other AAA module. The
signaling 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, 37, 38). Consistent with this model, N-domains are
not essential for oligomerization of ClpA or ClpB (23, 37, and this
work). The connector is apparently sterically inaccessible to proteases
because 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 (aa 1-353).
Similar findings have been reported for ClpA (37). The reported
consequences of N-terminal deletions of ClpB on its chaperone activity
are contradictory. Zolkiewski and co-workers (23) showed that a ClpB
variant, starting from the internal start site (aa 149-857), was
completely inactive in refolding of aggregated luciferase. In contrast,
a ClpB 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), because this deletion variant
interacted with casein and unfolded luciferase indistinguishable from
ClpB wild type (38). N-domains may therefore be involved in a mechanism
for coupling the ATPase cycle of ClpB with its proposed unfolding
activity. Because 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 is not involved in
thermotolerance (43). 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 (44) and the AAA protein p97 (45, 46).
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 suggested recently (47) for the
yeast homologue Hsp104. A comparison of different ClpB fragments (aa
1-409 and 1-567) with respect to their oligomerization and their
resistance to proteolysis supports this model. First, cross-linking
studies revealed that ClpB-(1-567) can form dimeric species in
contrast to the shorter variant (aa 1-409), which remains monomeric
under all conditions. Additionally, partial proteolysis revealed a more
pronounced protection of the full-length version (aa 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 (37). 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 (48), 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 full-length ClpB. We assume that the linker is not an
integral part of the ClpB hexamer, because oligomerization was still
possible in case of a ClpB variant missing the linker region
(ClpB-(
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical domain (C-domain). The ATP binding pocket is located at the
interface of neighboring 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 also been proposed to
mediate substrate interaction and were therefore termed the sensor and
substrate discrimination
(SSD)1 domains (6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(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 an SpeI site, leading to the insertion of two
amino acids (Thr and Ser) at the deletion site. Reinsertion of the
linker was obtained by PCR amplification of the corresponding region
(amino acid 410-530) and addition of an SpeI site 5' and an
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 (Thr and Ser or Ala and Ser) at each
domain boundary (ClpB
LBL). Each mutagenesis was confirmed by DNA sequencing.
clpB::kan cells (11). Purifications
of DnaK, DnaJ, and GrpE were according to published protocols (14).
Pyruvate kinase and
-casein were purchased from Sigma; malate
dehydrogenase (MDH) was from pig heart muscle, and firefly luciferase
from Roche Applied Science. Protein concentrations were determined with
the Bio-Rad Bradford assay using bovine serum albumin as standard. Protein concentrations refer to the protomer.
-32P]ATP (0.1 µCi, Amersham Biosciences). ATPase activities were also determined in
the presence of 0.25 mg/ml
-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
program GRAFIT version 3.0 (Erithacus software).
S).
Proteolysis was initiated upon addition of 0.2 µg/ml thermolysin or
subtilisin, and generated cleavage products were analyzed by SDS-PAGE
(15%) and silver staining. Kinetic analysis of the degradation
reaction revealed the occurrence of stable fragments after 30-60 min
of incubation time. Identity of cleavage products was determined by
N-terminal sequencing (TopLab) and mass spectrometry. For mass
spectrometry analysis, bands were excised from one-dimensional
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 previously (16)
using a QSTARTM Pulsar (MDS Sciex, Toronto, Canada)
equipped with a nanoelectrospray ion source (MDS Proteomics, Odense,
Denmark). Sequence searches were performed with the Protein and Peptide
Software Suite (MDS Proteomics).
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 afterward. Defects in protein disaggregation in
clpB::kan mutant cells were followed
by isolation of aggregated proteins as described (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 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 and ATP-2), which are separated by a
linker region, and a C-terminal SSD. Both nucleotide binding domains
contain the Walker A
(206GX4GKT213
and
605GX4GKT612)
and Walker B
(276Hy2DE279 and
675Hy2DE678) motifs,
where X = any amino (aa) and Hy = hydrophobic amino acids. Conserved Arg residues (R) present
in both nucleotide binding domains (Arg-332 and Arg-756) 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 boldface) were subjected
to alanine mutagenesis, as indicated by arrows.
N-(143-857) version. The C-terminal SSD was removed in the
ClpB
SSD variant (aa 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 AAA modules (2). Several ClpB deletion variants were
constructed to test this possibility: ClpB- (1-409),
ClpB-(551-857), and ClpB-(1-567).
and
nucleotides in the first and
second AAA-domain, respectively (19). The conserved Glu residues of the
Walker B motif (Glu-279 and Glu-678 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
-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).
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Fig. 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 (2 mM 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 the presence of ATP (2 mM) in the running buffer.
Oligomerization of ClpB fragments and mutants
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Fig. 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
(lane 2) or the presence of 2 mM ATP (lane
3) or ADP (lane 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 (lane 1) served as control.
B, ClpB deletion variants were incubated in the absence
(lane 2) or presence of 2 mM ATP (lane
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 (lane 1) served as control.
SSD
variant (aa 1-758) and a longer version of the first AAA module (aa
1-567) some dimeric and trimeric species were observed (Fig.
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 (Fig. 3A and Table I). 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) that misses the linker region.
This variant exhibited only a ladder of cross-linking products in the
absence of nucleotide and required ATP for full 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.
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Fig. 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 (gray 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)2SO4) (gray
dotted line). The maximum values 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)2SO4 (100 mM).
Cross-linked proteins were separated on SDS (4-12%)-polyacrylamide
gels, followed by silver staining.
S cleavage was further limited, and reduced
amounts of the smaller fragments were obtained (Fig.
5A).
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Fig. 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/ATP S for 60 min. Cleavage products were
separated on SDS (15%)-polyacrylamide gels, followed by silver
staining. 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 as follows: 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 corresponds to the core domains of the AAA modules.
C-1/2 represents 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.
SSD) or reduced stability of hexamers
(ClpB-(
410-532)) were degraded more quickly in the presence of ATP
compared with WT ClpB and thus did not exhibit stabilization of the
full-length version (Fig. 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
because the control construct (ClpB
LBL) with the reinserted linker
region exhibited the same reduced proteolytic stability. Isolated
AAA-domains (1-409 and 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 the
presence of ATP exhibited some protection from degradation. Further
structural analysis of ClpB point mutants surprisingly revealed that
conformational changes 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 were only partially observed in the case
of K611A and 813AAA815, ClpB mutants that
nevertheless showed stabilization of the full-length protein (Fig.
5C). Thus oligomerization protects full-length ClpB even in
case of mutants that do not bind nucleotide tightly at the second AAA
module (K611A; 813AAA815). We conclude that
stabilization of full-length 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
the ClpB mutants. Structural integrity with respect to oligomerization
and stability during partial proteolysis was only completely preserved
in Walker B mutants of ClpB, although Walker A and the sensor 2 mutants
exhibited significant structural deficiencies. Interestingly, the
double Walker B mutant (E279A/E678A) was much more resistant toward
proteolysis than the single Walker B mutants and ClpB wild type.
Because this mutant is deficient in ATP hydrolysis (see below), the
occurrence of the other fragments (aa 3-331 and 3-351 and 537-857
and 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 with the
situation where only one AAA-domain has ATP bound and the other domain
has ADP bound.
-casein (20-fold excess over ClpB monomers). ClpB
N had a slightly increased ATPase activity but was less stimulable by casein,
in agreement with published data (23, 28). Removal of the linker region
(ClpB-(
410-532)) strongly decreased the basal ATPase activity by
4-fold, although 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 (aa 1-409, 1-567, 551-857, and 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 Arg-332 were enhanced 2-3-fold in low salt
buffer, conditions that favor oligomerization (data not shown). In
agreement with these findings, addition of 100 mM
(NH4)2SO4 or 300 mM
NH4Cl diminished the ATPase rate of full-length ClpB or
ClpB
N 3- and 5-fold, respectively (Table
II, 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
toward high ionic strength conditions. Addition of 100 mM
(NH4)2SO4 strongly reduced the
ATPase activity of mutants, in which the second AAA-domain was
inactivated (K611A and E678A), whereas variants with only an active
second AAA module (E279A and K211A) were only partially affected (Table
II), 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).
ATPase activities of ClpB fragments and mutants
-casein and the factors of ATPase inhibition in the presence
of 100 mM ammonium sulfate are given. ND, not determined.
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Fig. 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 -casein (0.25 mg/ml).
Chaperone activities of ClpB mutants and fragments
clpB mutant cells (+, full activity; (
),
residual activity;
, no activity). ND, not determined.
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), because 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 full-length ClpB.
Full disaggregation activity of ClpB
N was also observed when the
ClpB concentrations in the activity assays were further reduced,
thereby increasing the dependence of the disaggregation on ClpB (data
not shown).
clpB mutant cells by IPTG induction of 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 with wild type cells (thermotolerance). We therefore tested
the ClpB variants for their ability to re-establish protein disaggregation and thermotolerance in
clpB mutant cells
(Fig. 7). Complementation studies were
performed in the presence of 25 µM IPTG, leading to
3-4-fold increased ClpB levels as compared with 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 (Fig. 7 and Table III). Constructs with partial
chaperone activities in vitro, such as E279A, also exhibited
some activity in vivo.
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Fig. 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.
Subsequently 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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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 stabilizing the ClpB hexamer. Several observations support this
model. First, the Walker A (K212A) and Arg finger (R332A) mutants of
the first AAA module did not form stable hexamers in the presence of
ATP, whereas corresponding mutations in the second nucleotide
binding domain (K611A and R756A) had no influence on ClpB
oligomerization, although these mutants exhibited severe structural
deficiencies. Second, the introduction of amino acids into the C-domain
of the first AAA module (ClpB-(
410-532) and 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 100 mM (NH4)2SO4) cause 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 (29). 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 Trp-543, 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 Trp-543 fluorescence reflect interactions of the first C-domain not only with itself but
also adjacent ATPase domains, thereby leading to increased shielding of
Trp-543 and stabilization of the oligomer.
Nucleotide-dependent conformational changes of the
C-terminal
-helical domain was also demonstrated by limited
proteolysis. Whereas the C-domain was rapidly degraded in the absence
of nucleotides, it became largely resistant to proteases upon
nucleotide addition (Fig. 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 adjacent subunits, thereby becoming
less accessible to proteases. Consistently, ClpB mutants with defects
in nucleotide binding (K212A and K611A;
813AAA815) did not exhibit stabilization of the
corresponding C-domains.
-casein, is most likely because of the stabilization of ClpB oligomers.
N-(143-857) variant showed the same chaperone activity as
wild type ClpB both in vitro and in vivo
(resolubilization of protein aggregates and 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 full-length ClpB;
likewise, the N-terminal truncated version of T. thermophilus ClpB was also shown to be active in protein
disaggregation in vitro (39, 40). 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 (41, 42).
410-532)). Similarly, the I-domain of HslU, although inserted into the AAA-domain, forms an independent 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. Because the
ATPase activity of ClpB-(
410-532) was still strongly stimulable 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 for coupling ATP hydrolysis and substrate
unfolding, as proposed recently by Lindquist and co-workers (47).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank D. Dougan and K. Turgay for discussions and critical reading of the manuscript. We also thank A. Schulze-Specking for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Bu617/14-1 and by the Fond der Chemischen Industrie (to B. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed. E-mail: a.mogk@zmbh-uni-heidelberg.de.
To whom correspondence may be addressed. E-mail:
bukau@zmbh.uni-heidelberg.de.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M209686200
2 J. Weibezahn, C. Schlieker, B. Bukau, and A. Mogk, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SSD, sensor- and
substrate discrimination;
DTT, dithiothreitol;
ATPS, adenosine
5'-O-(thiotriphosphate);
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
MDH, malate
dehydrogenase;
WT, wild type;
aa, amino acid.
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