From the
Interactions of the DnaK (Hsp70) chaperone from Escherichia
coli with substrates are controlled by ATP. Nucleotide-induced
changes in DnaK conformation were investigated by monitoring changes in
tryptic digestion pattern and tryptophan fluorescence. Using
nucleotide-free DnaK preparations, not only the known ATP-induced major
changes in kinetics and pattern of proteolysis but also minor
ADP-induced changes were detected. Similar ATP-induced conformational
changes occurred in the DnaK-T199A mutant protein defective in ATPase
activity, demonstrating that they result from binding, not hydrolysis,
of ATP. N-terminal sequencing and immunological mapping of tryptic
fragments of DnaK identified cleavage sites that, upon ATP addition,
appeared within the proposed C-terminal substrate binding region and
disappeared in the N-terminal ATPase domain. They hence reflect
structural alterations in DnaK correlated to substrate release and
indicate ATP-dependent domain interactions. Domain interactions are a
prerequisite for efficient tryptic degradation as fragments of DnaK
comprising the ATPase and C-terminal domains were highly
protease-resistant. Fluorescence analysis of the N-terminally located
single tryptophan residue of DnaK revealed that the known ATP-induced
alteration of the emission spectrum, proposed to result directly from
conformational changes in the ATPase domain, requires the presence of
the C-terminal domain and therefore mainly results from altered domain
interaction. Analyses of the C-terminally truncated DnaK163 mutant
protein revealed that nucleotide-dependent interdomain communication
requires a 15-kDa segment assumed to constitute the substrate binding
site.
The highly conserved Hsp70 proteins constitute a cellular
chaperone system for assistance and control of protein
folding(1, 2, 3) . This chaperone activity
relies on the ability of Hsp70 proteins to bind nonnative protein
substrates(4) , thereby preventing off-pathway substrate
folding, and to release these substrates upon ATP addition (5, 6),
thereby promoting further folding(4, 7) . ATP is
essential for virtually all Hsp70 chaperone functions(2) , and
mutations in the Escherichia coli DnaK homolog impairing
ATP-mediated substrate release render this chaperone inactive in
protein-folding reactions(8) . The ATPase activity of Hsp70
proteins is itself under tight control of cofactors, which thereby
regulate chaperone activity. In the case of DnaK, the DnaJ and GrpE
cofactors stimulate
Hsp70 proteins are composed of a highly conserved
N-terminal ATPase domain of 44 kDa and a C-terminal substrate binding
domain of 25 kDa (18-20) (Fig. 1). The three-dimensional
structure of the ATPase domain of the bovine Hsc70 homolog has been
resolved to 2.2-Å resolution(21) . It consists of two
quasisymmetric subdomains separated by a nucleotide binding cleft and
connected by two crossed helices. These helices are proposed to serve
as a hinge that mediates nucleotide-dependent opening and closing of
the nucleotide-binding cleft. ATP-induced changes in fluorescence of
the single tryptophan residue of DnaK located in the ATPase domain have
been interpreted as indication of such an ATPase subdomain movement
(22). The C-terminal domain consists of a well conserved 15-kDa
subdomain adjacent to the ATPase domain that probably comprises the
substrate binding site(18, 23, 25)
To investigate the mechanism of coupling between
ATPase activity and substrate interaction, we analyzed conformational
changes in DnaK induced by nucleotides and cofactors. This was achieved
by monitoring changes in proteolytic degradation and tryptophan
fluorescence of DnaK. Evidence is presented for major conformational
changes in the substrate binding region of DnaK induced by the binding
of ATP, and in the ATPase domain induced by ADP and ATP, respectively.
The detected ATP-induced conformational changes depended on the
interaction of the ATPase domain with the substrate binding region of
DnaK. The presented data provide an insight into the structural basis
of the coupling of ATPase activity with substrate binding.
Addition of ADP to
nucleotide-free DnaK did not accelerate proteolysis and did not cause
the major changes in proteolysis pattern found for ATP, as judged by
the lack of the 21- and 53-kDa fragments (Fig. 2, upperpanel). However, the fragment pattern obtained in
presence of ADP is distinct from the pattern of nucleotide-free DnaK in
that (i) the 14.5- and 18-kDa fragments disappeared, (ii) the 33- and
31-kDa fragments were less populated, and (iii) the 46-kDa fragment was
more populated (Fig. 2). These ADP-induced changes in the
proteolysis pattern of DnaK escaped previous detection, possibly
because DnaK preparations were used that contained significant amounts
of prebound ADP. Together, our data suggest that DnaK can adopt at
least three distinct conformational states that are determined by the
absence of nucleotide, bound ADP, and bound ATP.
In the absence
of nucleotides (data not shown), the kinetics and pattern of trypsin
digestion of DnaK were not detectably altered by the addition of DnaJ
and GrpE at relative stoichiometries of 1:10 and 1:1, respectively, or
of saturating amounts (10 µM) of a model substrate of
DnaK, peptide C of vesicular stomatitis virus glycoprotein(5) .
Failure to detect conformational changes in DnaK did not result from
premature tryptic digestion of these cofactors since increase in the
amounts of DnaJ and GrpE did not significantly change the results. In
contrast, in the presence of ATP, the addition of DnaJ changed the
degradation pattern of DnaK toward a pattern normally found in the
presence of ADP, i.e. disappearance of the 21-kDa fragment and
decrease in abundance of the 53 kDa fragment (Fig. 4). Since in
this experiment DnaJ was added to DnaK at substoichiometric (1:10)
amounts, DnaJ is unlikely to induce these conformational changes in
DnaK by complex formation. The most likely interpretation is that DnaJ,
which stimulates the DnaK ATPase activity by acceleration of the
rate-limiting step,
In the case of GrpE, the
addition of equimolar amounts of GrpE to nucleotide-free DnaK in the
presence of ATP did not cause significant changes in the tryptic
degradation pattern of DnaK (data not shown). This suggests that GrpE
does not induce major conformational changes in DnaK detectable by the
method used. It cannot be ruled out, however, that short-lived
interactions between GrpE and DnaK exist in the presence of ATP that
involve significant alterations in DnaK structure.
The 31- and 33-kDa
fragments enriched in the absence of ATP were recognized by mAb
K2-4 and therefore contain C-terminal sequences. Microsequencing
revealed that the N terminus of the 31-kDa fragment is at the border
between the ATPase domain and the C-terminal domain at residue Asp-388
and that the N terminus of the 33-kDa fragment is within the ATPase
domain at residue Lys-363 (Fig. 5B). The C termini of
both fragments are most likely at the authentic C terminus of DnaK
since the apparent molecular weights of both fragments were even higher
than those predicted for fragments of these sizes. Aberrant
electrophoretic mobility was indeed found for a polymerase chain
reaction-designed C-terminal fragment of DnaK, DnaK386-638 (Fig. 6).
The 21-
and 16-kDa fragments, enriched in the presence of ATP, originate from
trypsin cuts at residues Arg-467 and Arg-517, respectively. These
cleavage sites reside within the substrate binding region of Hsp70
proteins(18, 23) .
Taken together, these data indicate that
the major alterations in tryptophan fluorescence in response to ATP
addition result from alterations in the interaction of the ATPase
domain with the substrate binding domain, and not, as previously
thought, directly from the nucleotide binding event.
We used partial proteolysis pattern and intrinsic tryptophan
fluorescence to monitor nucleotide-induced conformational changes in
DnaK. We were able to demonstrate three different conformations of DnaK
specified by the absence of nucleotides and the presence of ADP and
ATP, respectively. Our data provide evidence for nucleotide-controlled
interactions of the ATPase domain with the substrate binding region
adjacent to the ATPase domain. These domain interactions are indicated
by the findings that (i) major nucleotide-induced tryptic cleavage
sites localize to the C terminus of DnaK, (ii) protease susceptibility
is strongly reduced for DnaK fragments comprising separately the 44-kDa
ATPase domain and the 25-kDa C-terminal domain, and (iii)
nucleotide-induced tryptophan fluorescence changes observed with
DnaK
The observed ADP-induced conformational changes occur within and at
the C-terminal end of the ATPase domain, resulting in protection of
trypsin cleavage sites at Arg-188, Arg-362, and Lys-387 (Fig. 9).
These changes seem to result from an altered interaction with the
C-terminal domain since addition of ADP to the nucleotide-free ATPase
domain fragment DnaK1-385 does not detectably affect its
proteolytic cleavage pattern (data not shown). Residues Arg-188 and
Arg-362 are located in close proximity at the surface of ATPase
subdomain IIa near the connecting hinge (Fig. 10), demonstrating
accessibility and flexibility of the area surrounding the hinge region
of the ATPase domain. It is important to note that ADP did not induce
conformational changes within the substrate binding region. This
correlates well with the failure of ADP to trigger substrate
release(6, 27, 40) .
In another approach to
determine the role of ATP hydrolysis in the induction of structural
changes in DnaK relevant to substrate release, we investigated by
limited proteolysis whether the nonhydrolyzable ATP analogues ATP
Further differences between ATP and ADP in imposing
conformational changes on DnaK were revealed by tryptophan fluorescence
measurements. ATP, but not ADP, induces a blue-shift of DnaK tryptophan
fluorescence (6, 22, 40). This observation, together with our finding
that ATP-induced changes in tryptophan fluorescence require the
presence of the substrate binding region of DnaK (Fig. 8),
indicates that these fluorescence changes result from conformational
changes in the substrate binding region of DnaK. The emission maximum
at 339 nm in the absence of ATP suggests a partially hydrophilic
environment of residue Trp-102 at subdomain Ib. In addition, a
structural model of the DnaK ATPase domain that is based on C
The
differences between the ADP- and the ATP-bound conformers of DnaK and
other Hsp70 proteins observed by us and described in the literature (6,
19, 22, 40) prompted us to hypothesize nucleotide binding to Hsp70 as a
two-step process. In a first step, contacts between ADP or ATP and the
ATPase domain are made, probably involving the adenine binding pocket
and phosphate binding loops from subdomains I and
II(21, 48) . These initial contacts cause conformational
changes in the ATPase domain as described in this work for the
ADP-bound conformer, possibly involving subdomain movement leading to
the closing of the nucleotide binding cleft(8, 48) . In
the case of ATP, a second step could involve correct positioning of the
Mg
Major structural elements
required for the observed interdomain interactions are located within a
stretch of 150 amino acids adjacent to the ATPase domain, which is
proposed to build up the substrate binding region of DnaK.
We thank H. Bujard for continuous support, M.
Büttner for excellent technical assistance, G. C. Walker for gift
of DnaK-T199A, A. Bosserhoff and R. Frank for microsequencing of DnaK
fragments, A. Valencia and C. Sander for providing a three-dimensional
model of the DnaK ATPase domain and for help with structural
interpretations, R. Mosbach and Y. Cully for help with the figures of
the three-dimensional model of the DnaK ATPase domain, and T.
Hesterkamp for critically reading the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-phosphate cleavage and nucleotide exchange,
respectively(9, 10) .
(
)The recent
identification of DnaJ and GrpE homologs in procaryotes and
eukaryotes(11, 12, 13, 14, 15, 16, 17) indicate
conservation in evolution of the E. coli DnaK-DnaJ-GrpE
chaperone system.
(
)and a less well conserved C-terminal 10-kDa subdomain
of unknown function (Fig. 1).
Figure 1:
Proteins used in this study.
DnaK is depicted as three-domain protein. The functions of the domains
are indicated at the top. The relevant residue numbers are
shown for each protein. DnaK-T199A has a threonine to alanine point
mutation at residue 199 that impairs its ATPase activity (32). DnaK163,
DnaK1-385, and DnaK386-638 are fragments comprising the
indicated domains of DnaK. DnaK386-638 was cloned as N-terminal
hexahistidine fusion protein for convenient
purification.
Little is known about the
mechanism of coupling between the cycle of substrate binding/release
and the ATPase activity. At a functional level, coupling is manifested
by the mutual stimulation of ATPase activity and substrate
release(5, 6, 8, 26) . Recent
experiments established that the mere binding of ATP is sufficient to
cause efficient substrate release from DnaK(6, 10) .
Hydrolysis of ATP converts DnaK from an ATP-bound form, characterized
by high on and off rates of substrate interactions, to an ADP-bound
form, characterized by low on and off rates(10, 27) .
The ATPase activity of DnaK thus constitutes a switch regulating the
velocity and stability of substrate binding by DnaK. At a structural
level, ATP-induced conformational changes in DnaK were detected by
means of ATP-induced changes in pattern of proteolysis and in infrared
and fluorescence spectra(22, 26) . A correlation between
hydrolysis of ATP, alteration in the degradation pattern, and release
of a prebound model substrate, bovine pancreas trypsin inhibitor, was
reported for DnaK(26) . The recent finding that the binding of
ATP is sufficient to trigger substrate release raises the question
whether it is also sufficient to trigger conformational changes in
DnaK, e.g. in the substrate binding region, which then induce
substrate release.
Protein Purification
DnaK,
DnaK1-386, DnaK163, and N-terminally hexahistidine-tagged
DnaK387-638 were purified as described (28) after
overproduction in
dnaK52 cells(29) . DnaJ and GrpE
were purified as described previously(30, 31) . The
DnaK163 mutant protein lacks the carboxyl-terminal 100 residues of DnaK
due to an amber mutation in the dnaK163 allele.
DnaK-T199A was purified as described previously (32).
Nucleotide-free preparations of DnaK were obtained by a procedure to be
published elsewhere.
(
)The absence of bound
nucleotides was verified by reversed phase high performance liquid
chromatography analysis using a C
column.
Proteolysis of DnaK
Partial tryptic
degradation of DnaK was performed as described previously(8) .
Briefly, DnaK (4.3 µM) was preincubated in buffer T (40
mM Tris-HCl, pH 7.6, 8 mM mg(OAc), 20
mM NaCl, 20 mM KCl, 0.3 mM EDTA, 2 mM DTT) (8) at 30 °C for 30 min in the presence or absence
of nucleotides (ATP, Sigma; ADP, Waldhof Chemie, Mannheim). Identical
degradation patterns were obtained after 5 min or 2 h of preincubation,
respectively. Proteolysis was started by the addition of 0.15 µg of
trypsin (Merck, Darmstadt). At various time points, aliquots were taken
and analyzed by SDS-polyacrylamide gel electrophoresis (33) or
16.5% Tricine
(
)step-gel electrophoresis (34) followed by silver staining (35) or immunoblotting
with monoclonal antibodies K3-1 or K2-4 using standard
protocols.
Monoclonal Antibodies
The monoclonal antibodies
K3-1 and K2-4 were obtained by standard methods from Balb/c
mouse spleen cells immunized with DnaK antigen and PAI myeloma cells.
DnaK-specific hybridomas were identified by enzyme-linked immunosorbent
assay and Western blot analyses. The antibodies were purified by
affinity chromatography on Protein G-Sepharose.
Microsequencing
DnaK (60 µg) was preincubated
with or without 5 mM ATP for 30 min at 30 °C. After the
addition of trypsin (1.3 µg), partial proteolysis was allowed to
proceed for 10 min (without ATP) and 4 min (with ATP), respectively,
and stopped by addition of phenylmethanesulfonyl fluoride (3
mM, final concentration) and 2 protein sample buffer.
The samples were immediately boiled and separated on 12.5% Laemmli and
16% Tricine step gels. The bands were electroblotted onto Selex PP
membrane (Schleicher and Schüll) with 10 mM CAPS
/NaOH, pH 11, 10% methanol blotting buffer. The
membranes were stained with naphthol blue black (Sigma, 0.1% (w/v) in
10% methanol, 2% acetic acid), and destained in 50% methanol, 7% acetic
acid. The fragments of interest were excised and sequenced on an ABI
protein sequencer model 473A using standard methodology.
Fluorescence Spectra
Measurements of the intrinsic
tryptophan fluorescence of DnaK were performed on a SML
``Smart'' 8100 photon-counting spectrofluorimeter.
Nucleotide-free DnaK (2 µM) was analyzed at 25 °C in a
buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl,
5 mM MgCl, 2 mM EDTA, and 2 mM dithioerythrol. Emission was recorded at a fixed excitation
wavelength of 290 nm. Spectra in the presence of ATP were recorded
after the addition of ATP from a concentrated stock solution (744
µM, final concentration) and 5-min incubation in the
cuvette. Under these conditions,
99% of the enzyme is saturated
with ATP, whereas
1% of the substrate has been turned
over(10) .
Limited Proteolysis of DnaK Reveals Distinct ATP- and
ADP-induced Conformational Changes
Limited proteolysis has been
demonstrated to be a useful tool for the detection of functional
domains and nucleotide-induced conformational changes in various Hsp70
proteins (18, 26, 36). On the basis of previous work, we extended
limited proteolysis of DnaK to further dissect the conformational
changes that are induced by nucleotides and cofactors. Compared with
published procedures, we introduced three changes that were crucial for
detection of previously undiscovered conformational changes. First, we
used as starting material a nucleotide-free preparation of DnaK instead
of standard preparations that presumably have nucleotides bound with a
stoichiometry of up to 1:1(37, 38) . Second,
SDS-polyacrylamide gels of proteolytic digests of DnaK were subjected
to highly sensitive silver staining according to Blum(35) ,
which allows detection of minor proteolytic fragments. Third, fragments
smaller than 20 kDa were identified by separation in a 16.5% Tricine
gel system(34) . Fig. 2shows the kinetics of partial
trypsin digestion of DnaK in the absence or presence of ATP and ADP
after separation of the degradation products by SDS-polyacrylamide gel
electrophoresis. In the absence of nucleotide, degradation was
relatively slow and yielded major fragments of 55, 46, 44, 33, 31, and,
as revealed by use of the 16.5% Tricine gel system, 18 and 14.5 kDa
apparent molecular masses (Fig. 2).
Figure 2:
Partial tryptic digest of DnaK in the absence and presence of nucleotide. Aliquots of a reaction
mixture containing DnaK and the indicated nucleotide (50
µM, final concentration) were taken at the indicated time
points and analyzed by gel electrophoresis using 12.5% Laemmli (upperpanel) and 16.5% Tricine step (lowerpanel) gels. The asterisks indicate the trypsin
band.
Addition of ATP
accelerated degradation significantly and changed the fragment pattern
in that (i) the 46-kDa fragment became by far the most prominent
product, (ii) the 33-, 31-, 14.5-, and 18-kDa fragments almost
completely disappeared, (iii) new 21- and 53-kDa fragments were
produced transiently, (iv) a 16-kDa fragment became much more
prominent. It is important to note that the apparent lack of the 21-
and 53-kDa fragments in the absence of ATP makes these fragments the
most intriguing indicators of ATP-induced conformational changes in
DnaK. The ATP-induced changes in kinetics and pattern of proteolysis of
DnaK described here are consistent with those reported by Liberek et al.(26) , except that the use of the Tricine gel
system resulted in better resolution of smaller fragments and allowed
us to identify additional fragments (14.5, 16, and 21 kDa) that appear
or disappear in dependence of ATP.
Binding of ATP Is Sufficient to Induce ATP-mediated
Conformational Changes in DnaK
The recent finding that ATP
hydrolysis is not needed for efficient substrate release by DnaK (6, 10) prompted us to investigate whether it is needed
for the ATP-induced conformational changes in DnaK described above. In
one approach we tested whether an ATPase-deficient DnaK mutant protein,
DnaK-T199A(32) , which has retained the ability to release bound
substrates upon ATP addition(6) , also exhibits the
conformational changes found in DnaK upon ATP
addition. For this purpose, we subjected the DnaK-T199A mutant protein
to partial trypsin digestion in the presence and absence of
nucleotides. The kinetics and the pattern of proteolysis of the
DnaK-T199A mutant protein were indistinguishable from those found for
DnaK
(Fig. 3). In particular, the 53-, 46-, 21-,
and 16-kDa fragments indicative of DnaK
in the
presence of ATP were also found for the mutant protein. This result
demonstrates that ATP hydrolysis is not essential for the ATP-induced
major conformational changes in DnaK.
Figure 3:
Partial
tryptic digest of the DnaK-T199A mutant protein in the absence and
presence of nucleotide. The DnaK-T199A mutant protein in the absence
and presence of nucleotides was subjected to partial tryptic
degradation as described in the legend of Fig. 2 and was analyzed by
gel electrophoresis using a 12.5% Laemmli
gel.
Effects of DnaJ, GrpE, and Peptide Substrates on DnaK
Conformation
The ATPase activity of DnaK is stimulated by
substrates(6, 8, 10) ,
DnaJ(9, 10) , and
GrpE(9, 28, 39) . We investigated the
possibility that the stimulating activity of these factors involves
induction of conformational changes in DnaK leading to alterations in
the kinetics and the pattern of proteolysis of DnaK.
-phosphate
cleavage(9, 10) ,
causes efficient
conversion of DnaK-bound ATP to ADP. This is consistent with the
finding that in the presence of DnaJ more than 95% of DnaK-bound
nucleotide is found as ADP under steady state conditions(10) .
It is important to note that our finding argues strongly against ATP
hydrolysis being required for the ATP-induced major conformational
changes in DnaK (26) and supports our results showing that it is
ATP binding that causes the observed significant conformational changes
in DnaK.
Figure 4:
Partial
tryptic digest of DnaK in the absence and presence of
ATP and DnaJ. Reaction mixtures containing DnaK
(4.3
µM), ATP (100 µM), and DnaJ (0.4
µM) where indicated were preincubated for 5 min at 30
°C prior to addition of trypsin (0 min), subjected to proteolysis,
and further analyzed by Laemmli gel electrophoresis as described in the
legend of Fig. 2. The arrowhead shows a major tryptic fragment
of DnaJ that is only slightly larger than the 21-kDa DnaK fragment (arrows) indicative of the ATP bound
conformation.
In the case of peptide C substrate, the addition of a
2-fold molar excess in the presence of ATP induced a conformational
change in DnaK that was qualitatively similar, but much weaker, to the
effect found with DnaJ (data not shown). This observed slight change in
DnaK conformation to an ADP-like form probably results from the limited
potential of peptide C to stimulate the ATPase activity of DnaK by
accelerating -phosphate cleavage(10) .
We
cannot, however, exclude that the proteolysis pattern is affected due
to direct protection of cleavage sites within the substrate binding
region of DnaK (see below) by peptide C.
Identification of Tryptic DnaK Fragments
To gain
insights into the structural basis of the nucleotide-induced
conformational changes of DnaK, we assigned the major tryptic fragments
to DnaK subdomains by immunological characterization and N-terminal
sequencing. For immunological characterization, monoclonal antibodies
with specificity for DnaK were prepared and used to identify fragments
carrying C- and N-terminal epitopes. We specifically used the
monoclonal antibodies (mAb) K3-1 and K2-4, which recognize
epitopes in the ATPase domain and the proposed C-terminal substrate
binding region (within 153 amino acids immediately following the ATPase
domain), respectively. These epitope specificities were determined by
immunoblot analysis using fragments of DnaK comprising the ATPase
domain (DnaK1-385; recognized by K3-1) and the C-terminal
domain (DnaK386-638; recognized by K2-4) and the DnaK163
mutant protein lacking the carboxyl-terminal 100 residues (recognized by K2-4 and K3-1) (Fig. 5A).
Figure 5:
Identification of major tryptic DnaK
fragments. A, characterization of the DnaK-specific monoclonal
antibodies K3-1 and K2-4. Purified DnaK,
DnaK163, DnaK1-385, and DnaK386-638 proteins (1 µg of
each) were loaded onto a 12.5% Laemmli gel, electroblotted on
nitrocellulose membrane, and immunodetected with the monoclonal
antibodies K3-1 (upperpanel) and K2-4 (lowerpanel). B, identification of DnaK
fragments by immunological analysis and microsequencing. Preparative
amounts of DnaK were partially digested by trypsin in the absence
(-) and presence (+) of excess ATP. The majority of the
samples was separated on 12.5% Laemmli and 16% Tricine step gels and
electroblotted onto polypropylene membrane for microsequencing (not
shown). Small aliquots of the samples were separated on 12.5% Laemmli
gels and analyzed by immunoblotting using monoclonal antibodies
K3-1 and K2-4 or by silver staining of the gel. The amino
acid sequences including the residue numbers of the N-terminal amino
acids of the corresponding bands are shown. The double band of
14.5 kDa was not separated after electrotransfer and therefore
resulted in two sequences starting with Thr-189 and Lys-363,
respectively.
Based on previous work on proteolysis of
Hsc70 (18) we expected that the 46-kDa fragment, which is
increased in abundance in the presence of ATP, comprises the ATPase
domain of DnaK, and that the 55- and 53-kDa fragments share this domain
and protrude carboxyl-terminally to different extents into the
substrate binding region. We confirmed this assumption by immunoblot
analysis and microsequencing (Fig. 5B). All three
fragments were recognized by the ATPase domain-specific mAb K3-1
and start with the second amino acid of the N-terminal sequence of
DnaK. The N-terminal formylmethionine residue of DnaK is apparently
cleaved off in vivo. Interestingly, the mAb K2-4
recognizes the 55-kDa fragment of DnaK but not the 53-kDa fragment,
indicating that its epitope is located near the C-terminal end of the
substrate binding domain around residue 500. Note that the prominent
46-kDa fragment is slightly larger than the 44-kDa ATPase domain of
DnaK, DnaK 1-385, that we constructed by polymerase chain
reaction-based cloning of the part of dnaK that encodes the
DnaK equivalent of the Hsc70 ATPase domain structure(21) . Since
the C-terminal domain starts near the minor trypsin cleavage site
Lys-387 (see below), the 46-kDa fragment is most likely the product of
a major tryptic cut at the first cleavage site in the C-terminal domain
at Lys-414. According to secondary structure prediction using the PHD
program(42, 43) , this residue is located in an
unstructured/loop region (data not shown).
Figure 6:
Partial tryptic digest of the
DnaK1-385 and DnaK386-638 proteins. The DnaK1-385 and
DnaK386-638 proteins comprising the ATPase and the substrate
binding domain of DnaK, respectively, were digested in the absence and
presence of ATP (100 µM, DnaK1-385) and peptide C
(10 µM, DnaK386-638) as indicated and analyzed by
gel electrophoresis using a 12.5% Laemmli gel as described in the
legend to Fig. 2.
The 18- and 14.5-kDa fragments, which occur mainly
in the absence of nucleotide, possess the same N termini as the 33- and
31-kDa fragments, respectively (Fig. 5B). Their smaller
sizes therefore resulted from additional tryptic cuts within the
C-terminal domain. This is also indicated by the failure of both mAbs
to recognize the 18- and 14.5-kDa fragments (Fig. 5B).
N-terminal sequencing revealed that a second fragment comigrated with
the 14.5-kDa fragment that has the N terminus within the ATPase domain
of DnaK at Thr-189. The silver-stained gel in Fig. 5B shows that indeed a doublet band at about 14.5 kDa exists that
could not be resolved after blotting for microsequencing. The relative
abundance of the upper band decreased upon the addition of nucleotide,
while the abundance of the lower band was not affected by nucleotides.
The nucleotide-dependent 14.5-kDa band is probably the 14.5-kDa
fragment starting with Thr-189 as this fragment yielded significantly
stronger microsequencing signals in the absence of nucleotide compared
with the fragment starting with Lys-363 (data not shown).
From the apparent sizes
of these fragments, we assume that their C termini correspond to the
authentic C terminus of DnaK. These ATP-induced conformational changes
in the substrate binding region demonstrate structural coupling between
the ATPase domain and the substrate binding domain of DnaK. To our
knowledge, this is the first evidence that nucleotide binding to the
ATPase domain of DnaK induces an altered conformation in the substrate
binding region. It is tempting to speculate that the ATP-induced
increased accessibility for trypsin digestion in the substrate binding
domain indicates those conformational changes that are responsible for
accelerated substrate release.
Proteolytic Susceptibility of the Substrate Binding
Domain of DnaK Requires the ATPase Domain
The trypsin digestion
experiments indicating ATP-dependent DnaK domain interactions led us to
speculate that these interactions are a prerequisite for the
proteolytic susceptibility of the DnaK domains. This possibility was
investigated by testing the accessibility of trypsin cleavage sites in
a C-terminal fragment of DnaK, DnaK385-638, and an ATPase domain
fragment, DnaK1-385 (Fig. 6). Both fragments were far more
resistant to proteolysis than DnaK, both in presence
and absence of ATP or peptide (compare patterns after 40 min of
incubation in Fig. 2and 6). In the DnaK385-638 fragment,
the only prominent cleavage event occurred rapidly and removed the
recombinant N-terminal hexahistidine tag, as revealed by
microsequencing of the resulting band (data not shown). It is important
to note that the minor cleavage products of DnaK385-638 did not
include the 21-kDa proteolytic fragment indicative of the ATP-bound
conformation of DnaK
. We therefore conclude that an
ATP-dependent interdomain interaction of the ATPase domain with the
substrate binding domain is required for proteolytic susceptibility of
the substrate binding region (in particular for cleavage at Arg-467).
This interdomain interaction could not be restored by mixing equimolar
amounts of the DnaK385-638 and DnaK1-385 fragments,
suggesting that intramolecular coupling is required (data not shown).
Our findings are consistent with the observation that these DnaK
fragments, when combined, did not exhibit chaperone activity in
DnaK-dependent refolding of firefly luciferase.
(
)
ATP-induced Conformational Changes in a DnaK
Mutant Protein Lacking the C-terminal 100 Amino Acids
While the
experiments described above allowed identification of a region within
the substrate binding domain of DnaK that is a target for ATP-induced
conformational changes, they did not identify the region within the
C-terminal domain that is required to mediate these changes. To obtain
information about this region, we tested whether the mutant protein
DnaK163, which lacks the 100 C-terminal amino acids of DnaK due to an
amber mutation in the dnaK163 allele, shows
ATP-induced conformational changes (Fig. 7). This mutant protein
has retained the ability to bind substrates and possesses wild-type
ATPase activity,
and therefore has maintained structural
integrity as judged by these functions. The DnaK163 mutant protein
showed a tryptic digestion pattern that was similar to that of
DnaK
with respect to some major fragments (e.g. the 55- and 46-kDa fragments) but was different with respect to
some other fragments, as expected from the lack of 100 C-terminal amino
acids (Fig. 7). Most importantly, as for DnaK
,
the degradation pattern and kinetics of the DnaK163 mutant protein
changed in dependence of ATP (Fig. 7). In particular, two
fragments of 13 and 15 kDa, respectively, were enriched in the absence
of nucleotide and disappeared upon the addition of ATP. No ATP-induced
new bands could be observed with DnaK163, potentially because the
expected small fragments are structurally unstable and rapidly further
degraded. With regard to degradation kinetics of the DnaK163 mutant
protein, the first visible cleavage event resulting in the 55-kDa
fragment occurred later in the presence of ATP than in the absence of
nucleotide, in contrast to DnaK
. This indicates lower
accessibility of the respective cleavage site in DnaK163 compared with
DnaK
.
Figure 7:
Partial tryptic digest of the DnaK163
mutant protein in the absence and presence of nucleotide. The
proteolysis products of the C-terminally truncated DnaK163 mutant
protein in the absence and presence of ATP were analyzed by gel
electrophoresis using 12.5% Laemmli (upperpanel) and
16.5% Tricine step (lowerpanel) gels as described in
the legend to Fig. 2. The asterisk indicates the trypsin
band.
The existence of ATP-induced conformational
changes in the DnaK163 mutant protein suggests that a key mechanism of
interdomain coupling is preserved in this protein and that this
mechanism consequently involves a stretch of 153 amino acids adjacent
to the ATPase domain. However, the difference that exists between the
DnaK163 mutant protein and DnaK with respect to the
kinetics of proteolysis suggests some additional role for the
C-terminal 100 amino acids of DnaK in the coupling process.
ATP-induced Changes in Tryptophan Fluorescence of DnaK
Require the Substrate Binding Domain
DnaK possesses a single
tryptophan residue (Trp-102) located in the ATPase subdomain Ib (see Fig. 10). ATP addition leads to both quenching and blue shift of
the tryptophan fluorescence emission
signal(6, 22, 40) . Identical changes in
fluorescence upon ATP addition were reported for the ATPase-deficient
DnaK-T199A mutant protein(40) , indicating that ATP binding is
sufficient to induce these changes. Fluorescence changes were
interpreted to result directly from conformational changes in the
ATPase subdomain following nucleotide binding(22) .
Figure 10:
Model of the three-dimensional structure
of the DnaK ATPase domain. The model is based on the C coordinates of the Hsc70 ATPase domain (21). The side chains were
oriented using an energy minimization program. The locations of
residues Trp-102 (W102), Arg-188 (R188), and Arg-362 (R362) are indicated. A, standard view. B,
side view demonstrating the surface exposure of the Trp-102 side
chain.
In view
of our data showing a requirement for interdomain coupling for
efficient proteolytic degradation of DnaK, we hypothesized that the
ATP-dependent tryptophan fluorescence changes in DnaK also result from
changes in the interaction of the ATPase domain with the substrate
binding region. This possibility was tested by recording the tryptophan
fluorescence emission spectra of nucleotide-free preparations of
DnaK, DnaK163, and DnaK1-385 before and after
the addition of ATP (Fig. 8). DnaK
and the
DnaK163 mutant protein both showed the typical ATP-induced alterations
of the fluorescence emission spectrum consisting of a blueshift of 4 nm
and a quench of
11% (Fig. 8, A and B). Note
that at the conditions used, more than 95% of DnaK-bound nucleotide is
ATP(10) .
(
)In agreement with a previous
publication(40) , emission spectra for the DnaK-T199A mutant
protein in the presence and absence of ATP were found indistinguishable
from those of DnaK
and DnaK163 (data not shown). We
conclude that the ATP-induced changes in both DnaK proteolytic
degradation and tryptophan emission spectrum result from binding, not
hydrolysis of ATP and that the C-terminal 10-kDa subdomain is not
involved in the alterations in tryptophan fluorescence.
Figure 8:
Tryptophan fluorescence spectra of
DnaK, DnaK163, and DnaK1-385. Emission spectra
of tryptophan fluorescence of DnaK
(A),
DnaK163 (B), and DnaK1-385 (C) at
concentrations of 2 µM in the absence (uppercurve) and presence (lowercurve) of
ATP (744 µM) were recorded between 310 and 370 nm at a
fixed excitation wavelength of 290 nm. The emission maxima without/with
ATP were 339/335, 338/334, and 339/338 nm for DnaK
,
DnaK163, and DnaK1-385, respectively. The fluorescence quenches
at 339 nm were 14, 11, and 5% for DnaK
, DnaK163, and
DnaK1-385, respectively.
In the
absence of ATP, the ATPase domain fragment, DnaK1-385, showed an
emission spectrum very similar to DnaK. In contrast to
DnaK
, however, the blueshift is almost completely
missing after the addition of ATP, with a residual shift of only 1 nm,
which is close to the recording accuracy, and the quenching is
decreased to 5% (Fig. 8C). As for DnaK
,
more than 95% of the DnaK1-385-bound nucleotide is ATP under the
conditions used.
Hypothetically, the observed fluorescence
changes could be assigned to alterations of the oligomeric state of
DnaK(31) , which is known to be nucleotide-dependent (24).
(
)For DnaK1-385, oligomerization cannot account
for the observed tryptophan fluorescence as it is a monomer under all
conditions tested.
For DnaK
, the
hypothesis of a role for deoligomerization in tryptophan fluorescence
changes was falsified, as after a 100-fold dilution of DnaK
into the fluorescence cuvette the fluorescence signal in absence
of ATP remained constant for a period of 2 h. These conditions are
known to allow deoligomerization of DnaK
(31) .
The spectra were recorded at DnaK concentrations (2 µM)
that produce far less than 10% oligomers of total
DnaK
(31) , and tryptophan fluorescence spectra
of DnaK
recorded at concentrations of 2 and 5
µM were identical, which is not consistent with the
hypothesis of a concentration-dependent dynamic equilibrium of
oligomeric states of DnaK(31) . Normalization of the tryptophan
emission spectra of DnaK
and DnaK1-385 in the
absence of ATP resulted in absolute identical spectra, making the
oligomeric state of DnaK very unlikely to influence its intrinsic
tryptophan fluorescence.
are strongly reduced for the ATPase domain
fragment of DnaK. The two methods used to monitor conformational
changes in DnaK are thus indicative for interdomain communication.
Figure 9:
Map of accessible tryptic cleavage sites
of DnaK in the absence and presence of ATP. DnaK is depicted as
three-domain protein. The tryptic cleavage sites in the absence (upperpart) and presence (lowerpart) of ATP as identified by microsequencing (cf. Fig. 5B) are indicated by their residuenumbers and arrows. The relative cleavage
frequency at a given site as judged by the abundance of the
corresponding protein fragment bands in gels (based on visual
inspection) is indicated by the thickness of the corresponding arrow. The map is drawn to scale, illustrating the
accumulation of accessible cleavage sites between residues 360 and 520.
Fast proteolytic cleavage of the C terminus, which slightly reduces the
size of DnaK, might have been undetected.
The conformational
changes induced by ATP include those that were detected upon the
addition of ADP. No additional ATP-induced structural alterations were
detected within the ATPase domain, although they are likely to occur.
The most prominent changes uniquely induced by ATP take place in the
C-terminal domain of DnaK, as indicated by the appearance of three
major tryptic cleavage sites at Lys-414, Arg-467, and Arg-517 (Fig. 5B). These three sites map to the beginning, the
center, and the end, respectively, of the proposed substrate binding
region of DnaK (Fig. 9). The existence of ATP-induced
conformational changes throughout the substrate binding region suggests
extensive structural reorganization of this subdomain rather than a
small, locally restricted motion of key residues. Consistent with this
idea is that the secondary structure prediction program PHD, which has
a very high (>70%) overall three-state
accuracy(42, 43) , predicts the substrate binding region
to have little
-sheet and
-helix contents and, therefore,
high structural flexibility. To our knowledge, this is the first report
of ATP-induced structural changes within the substrate binding region.
These changes are presumably correlated to the events leading to
substrate release. This latter assumption is further supported by
observation of a tight coupling between the events leading to
conformational changes in the substrate binding region (this study) and
to release of DnaK-bound substrates(6, 10) . Both
processes are triggered by binding, not hydrolysis, of ATP. They occur
not only in DnaK
but also in the DnaK-T199A mutant
protein defective in ATP hydrolysis.
S
and AMP-PNP induce conformational changes in DnaK. The trypsin
digestion patterns of DnaK in the presence of these analogues were
similar to the patterns found in the presence of ADP (data not shown).
In the case of ATP
S, however, no valid conclusion could be drawn
since the ATP
S preparation used (Fluka, Neu-Ulm) contained up to
25% ADP, which, at the conditions used, might saturate DnaK with ADP.
In the case of AMP-PNP, the ADP-like pattern can be explained by
assuming that it is not a valuable ATP analogue and imposes an
ADP-bound conformation on DnaK. This is indicated by the fact that
AMP-PNP binds to Hsp70 with a 100-fold lower affinity than ATP and
ADP(6, 49) . AMP-PNP might be unable to induce an
ATP-like conformation of DnaK because it cannot be coordinated
correctly at the active site (see below). Furthermore, the failure of
ATP analogues to induce ATP-like conformational changes in DnaK
correlates well with their failure to induce ATP-like fluorescence
changes(6)
and to release prebound
substrates(6) . Our findings raise doubts on the usefulness of
these ATP analogues to mimic the effects of ATP binding on DnaK
structure.
coordinates of the Hsc70 ATPase structure (21) and an
energy minimization program for the side chain conformations predicts
the tryptophan side chain to point outside of the ATPase domain (Fig. 10), which is unusual for that hydrophobic residue. It is
tempting to speculate that residue Trp-102 is directly involved in
interactions with the substrate binding region. Our idea is supported
by the fact that subdomain 2 of actin, the structural correspondent of
Hsp70 subdomain Ib, participates in several protein-protein
interactions, including contacts between actin monomers in the actin
filament (44) and the interaction with
-actinin (45) and fimbrin(46, 47) . An important feature
of actin polymerization into a filament is a rotation of subdomain 2 to
allow the contact to another actin monomer(44) . In the crystal
structure of the Hsc70 ATPase domain, subdomains Ib and IIb exhibit the
highest temperature factors, indicating flexibility of these
structures, too(48) . It will require further experimentation to
determine whether a movement of subdomain Ib is involved in the
transfer of conformational changes between the ATPase domain and the
substrate binding domain of Hsp70 proteins, including DnaK.
ATP complex at the active site in a highly coordinated
manner(48) . As a result of this very specific coordination of
Mg
ATP, further conformational changes in the ATPase domain occur
that were not detected by the approaches used. These changes affect
interdomain interactions with the substrate binding domain and induce a
low affinity state for substrates.
This requirement is indicated by the finding that the DnaK163
mutant protein lacking 100 C-terminal residues shows
nucleotide-dependent conformational changes in the ATPase domain and
the adjacent substrate binding region. The interactions found in
DnaK163 are sufficient to establish ATP-controlled substrate binding
and peptide-stimulated ATPase activity
and are therefore
relevant for chaperone activity of DnaK. We cannot exclude, however,
that the C-terminal 100 residues of DnaK that are missing in the
DnaK163 mutant protein also play a role in interdomain interactions
that remained undetected. Elucidation of the structure of the
C-terminal domain of DnaK will be required to understand the structural
basis for substrate binding and its coupling to the ATPase activity.
S, adenosine 5`-O-(thiotriphosphate); AMP-PNP,
5`-adenylyl-
,
-imidodiphosphate; mAb, monoclonal antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.