From the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932
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ABSTRACT |
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Recent reports have shown that the binding of ATP
to a 70-kDa molecular chaperone induces a rapid global conformational
transition from a "high affinity" state to a "low affinity"
state, where these states are defined by tight and weak binding to
(poly)peptides, respectively. To complete the activity cycle, a
chaperone molecule must ultimately return to the high affinity state.
In this report, this return to the high affinity state was studied
using a chemical cross-linking assay in conjunction with
SDS-polyacrylamide gel electrophoresis. The basis for this assay is
that in the absence of nucleotide or in the presence of ADP, conditions
that stabilize the high affinity state, cross-linking of the
Escherichia coli molecular chaperone DnaK yielded two
monomeric forms, with apparent molecular masses of 70 kDa (77%) and 90 kDa (23%), whereas cross-linking yielded only the 70-kDa monomeric
form in the presence of ATP. This ATP-dependent difference
in cross-linking was used to follow the kinetics of the low affinity to
high affinity transition under single turnover conditions. The rate of
this transition (kobs = 3.4 (±0.6) × 104 s
1 at 25 °C) is almost identical to
the reported rate of ATP hydrolysis (khy = 2.7 (±0.7) × 10
4 s
1 at 22 °C). These
results are consistent with a two-step sequential reaction where
rate-limiting ATP hydrolysis precedes the conformational change. Models
for the formation of two cross-linked DnaK monomers in the absence of
ATP are discussed.
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INTRODUCTION |
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The highly conserved and ubiquitous 70-kDa family of molecular chaperones, which include stress induced (Hsp70)1 and constitutively expressed (Hsc70) variants, promote protein-protein interactions via the of coupling ATP binding and hydrolysis to selective substrate binding and release. Molecular chaperones use this activity cycle to perform diverse biological processes such as the stabilization of partially unfolded and nascent proteins, protein translocation, and protein complex assembly and disassembly under normal growth and stress conditions (for reviews, see Refs. 1-3). The underlying molecular events that enable the coupling of ATP binding and hydrolysis to selective substrate binding and release are poorly understood.
The three-dimensional structure of the two separate 70-kDa chaperone
functional domains have been determined. The NH2-terminal domain of Hsc70 binds nucleotide in the base of a cleft formed by two
subdomains (4). Two K+ ions and a Mg2+ ion are
cofactors in the ATPase reaction by interacting with nucleotides in
this cleft (5). The requirement for K+ is specific, because
when K+ is replaced by Na+, (i) the rate of
Hsc70-catalyzed ATP hydrolysis is five times slower (6), and (ii)
DnaK-protein complexes do not dissociate in the presence of ATP (7).
The COOH-terminal domain of DnaK (residues 389-607) binds a peptide
substrate in a channel formed by the loops from a -sandwich (8). The
molecular mechanism which couples the activities of the two domains is
not understood.
Several lines of evidence indicate that ATP induces a global structural
transition in a 70-kDa chaperone molecule from a high affinity state to
a low affinity state (9-13). The high affinity state tightly binds
(poly)peptides, whereas the low affinity state weakly binds
(poly)peptides. Significantly, the induction of this structural
transition is a consequence of ATP binding and not hydrolysis, because
the rate of the transition (0.7 s1) (14) is much faster
than chaperone-catalyzed ATP hydrolysis (0.003-0.0005
s
1) (7, 15, 16). It appears that ATP hydrolysis is
involved in the reverse transition, that is, the return from the low
affinity state to the high affinity state (12, 14, 17). Small-angle x-ray scattering experiments conducted on recombinant bovine Hsc70 have
indicated that the reverse reaction occurs in at least two steps, where
the hydrolysis of ATP is followed by rate-limiting product release
(14).
In this report, a chemical cross-linking/SDS-PAGE assay was used to visualize the slow transition from the low affinity state to the high affinity state of the Escherichia coli 70-kDa molecular chaperone DnaK under single turnover conditions. The results are consistent with a two-step sequential reaction where ATP hydrolysis is rate-limiting rather than product release, and ATP hydrolysis induces a second reaction, a structural transition in the chaperone molecule. This assay can also be used to probe the effect of other parameters such as salt, temperature, and even cochaperones, on the kinetics of the transition.
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EXPERIMENTAL PROCEDURES |
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DnaK Purification--
DnaK was isolated from the E. coli strain RLM893 (18) as described previously (19). The protein
was maintained in a K+/HEPES sample buffer (25 mM HEPES, 50 mM KCl, 5 mM
MgCl2, 5 mM 2-mercaptoethanol at pH = 7.0)
and stored at 4 °C prior to use. Protein concentration was
determined by the Bio-Rad assay using bovine serum albumin as a
standard and verified spectrophotometrically (280 = 15.8 × 103 M
1
cm
1) (20). SDS-PAGE analysis demonstrated that the DnaK
preparations were >95% pure. For some of the cross-linking and
fluorescence experiments DnaK was dialyzed into a Na+/HEPES
sample buffer (25 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol at
pH = 7.0). All reagents were purchased from Sigma.
Cross-linking/SDS-PAGE-- Samples of DnaK were cross-linked for 2 min at 25 °C with glutaraldehyde (final concentration: 11 mM), and the reaction was stopped by the addition of an excess of glycine (final concentration: 200 mM). Cross-linked samples were loaded onto either a 4-12% linear gradient denaturing polyacrylamide gel or a 10% denaturing polyacrylamide gel, electrophoresed, and stained with Coomassie Brilliant Blue G-250. Gel lanes were scanned with a Molecular Dynamics densitometer to determine the relative amount of species in each lane.
Fluorescence Measurements--
A Photon Technology Inc. (South
Brunswick, NJ) StrobeMaster lifetime spectrometer with a SE-900 steady
state fluorescence option was used to monitor the slow increase in
tryptophan fluorescence in the single turnover experiments. In these
experiments the instrument was used in a time base mode, with
ex = 295 (3-nm bandwidth) and
em = 340 nm
(5-nm bandwidth). Samples were maintained in a quartz cuvette (1-cm
path length) with constant stirring and with temperature control via an
external circulating heating/cooling bath (
T = ± 0.2 °C). Sample temperature was verified using a hand held
thermocouple, which was placed directly into the sample.
Data Analysis and Reproducibility of Measurements-- The equilibrium and kinetic data were fit to the equations in the text using the program KaleidaGraph (Synergy, Reading, PA). All experiments were repeated three to four times.
The value for the equilibrium dissociation constant for ATP binding to DnaK has an error of 20%, while the values for the kinetic constants have 5 ![]() |
RESULTS |
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In the course of conducting cross-linking experiments to determine the conditions that promote the oligomerization of DnaK (21), we have noticed that the treatment of DnaK (20 µM) with glutaraldehyde produces two distinct monomer bands as well as dimer and trimer bands (Fig. 1A). On a 4-12% polyacrylamide denaturing gel, one monomer band is centered at a molecular mass of 70-kDa, as expected, while the upper monomer band is centered at an apparent molecular mass of 90-kDa. The broad protein bands are probably due to variations in the amount of intramolecular cross-linking (22, 23). Previously, two or more chaperone monomer bands have been reported and were attributed to heterogeneous cross-linking (22, 24). In this report, the occurrence of these two monomer bands was exploited to visualize ATP-induced conformational transitions in DnaK.
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Effect of Nucleotide on Cross-linking--
Fig. 1B
shows the effect of nucleotides on the cross-linking of DnaK in the
monomer region. At the concentration of DnaK (6 µM) used
in these experiments, 85-90% of the protein is monomeric (21). Since
10% polyacrylamide gels gave a slightly better separation of the two
monomer bands, 10% gels were used in these
experiments.2 Uncross-linked
DnaK appeared as a single monomer band (lane 1), whereas
DnaK that was cross-linked in the absence of nucleotide appeared as two
monomer bands (lane 2). When DnaK was cross-linked in the
presence of ADP (2.0 mM), the two monomer bands were still present (lane 3). In contrast, cross-linking in the presence
of ATP (2.0 mM) abolished the upper monomer band
(lane 4). Other nucleotide triphosphates, GTP, CTP, and UTP,
also abolished the upper monomer band (data not shown). On the other
hand, cross-linking in the presence of AMP-PNP (2.0 mM), a
nonhydrolyzable analog of ATP, had no effect on the upper band
(lane 5). On the basis of these results, we conclude that
the abolition of the upper monomer band is a consequence of a highly
specific conformational change in DnaK, induced by the action of
hydrolyzable nucleotide triphosphates. The -phosphate group
-O-P
O32
of the
nucleotide triphosphate is required for the induction of the
conformational change since the nucleotide triphosphate AMP-PNP, where
the
-phosphate group is
-NH-P
O32
, did not
induce the conformational change. Possibly the nitrogen atom linkage in
AMP-PNP modifies the coordination of Mg2+, which in turn
prevents the proper docking of AMP-PNP within the ATP binding site of
DnaK.
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Chromatography-- If Model 2 is correct it should be possible to separate the E state isomers. In an attempt to separate the putative isomers, DnaK was electrophoresed on both a 10-15% and a 15-20% linear gradient nondenaturing polyacrylamide gel, and, in each case, a single band was observed. Although unsuccessful in our attempts to separate these putative isomers, Model 2 should not be ruled out, as discussed below.
Equilibrium Experiments--
The thermodynamics of ATP binding to
DnaK were investigated by exploiting the different cross-linking
patterns of DnaK in the absence and presence of ATP. Samples of DnaK
(6.0 µM) with varying amounts of ATP were incubated for 1 min at 25 °C and then cross-linked. Fig.
3A shows the relative amounts
of the two monomer bands before and after the addition of ATP. The
upper monomer band was abolished when [ATP]/[DnaK] 1.6, indicating that the population of the E state was also
abolished.
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(Eq. 1) |
Single Turnover Experiments--
Since the upper monomer band is
abolished at near stoichiometric concentrations of ATP, the assay is an
ideal way to monitor the kinetics of structural transitions in a DnaK
monomer that occur during a single turnover. Single turnover
experiments at 25 °C were conducted by adding a stoichiometric
amount of ATP (6.0 µM) to a solution of DnaK (6.0 µM) in a K+/HEPES buffer (Fig.
4A). Aliquots were removed at
the indicated times and cross-linked as outlined under "Experimental
Procedures." Prior to the addition of ATP to DnaK, the two monomer
bands were present (lane 1), indicative of the E
state. Two minutes after the addition of ATP, the upper monomer band
was abolished (lane 2). The slow reappearance of the upper
band is shown in lanes 3-9. The lanes were scanned with a
densitometer to determine the relative amounts of the two DnaK
monomers, and the results are plotted in Fig. 4B. The
reappearance of the signal from the upper monomer band followed single
exponential kinetics according to S(t) = A(1 e
kobst) + B, with kobs = 3.4 (±0.6) × 10
4 s
1. Significantly, this rate is
identical within experimental error to the reported steady state rate
of DnaK-catalyzed ATP hydrolysis (khy = 2.7 (±0.7) × 10
4 s
1 at 22 °C) (25). On the
basis of these gel kinetic results, we conclude that (i) the
ATP-induced transition from the high affinity state to the low affinity
state is fast (t1/2 < 2 min) in the presence of
K+ ions, therefore this transition is due to ATP binding
and not hydrolysis; and (ii) the reverse transition (E**
E) occurs at the same rate as DnaK-catalyzed ATP
hydrolysis.
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DISCUSSION |
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The gel- and fluorescence-detected single turnover experiments
showed that ATP induces a rapid conformational change in a molecule of
DnaK. For example, the addition of a stoichiometric amount of ATP to
DnaK lead to the elimination of the upper monomer band (Fig.
4A) within 2 min or less. Similarly, the addition of a
stoichiometric amount of ATP to DnaK resulted in a rapid reduction in
fluorescence (Fig. 5). Since the rapid conformational change and the
rapid spectral change occur much faster than the steady state rate of
ATP hydrolysis (25), and because no rapid burst phase of ATP hydrolysis
has been detected in any single turnover experiments (7), we conclude
that the conformational change and the attending spectral change are
due to ATP binding to DnaK. This conformational change is consistent
with the high affinity state to low affinity state transition
(E E**) (7, 15, 16).
The major finding in this study was that the gel-detected
conformational change and the change in tryptophan fluorescence occur
at exactly the same rate (3 × 104
s
1), the rate of DnaK-catalyzed ATP hydrolysis. The
simplest explanation of these results is that ATP hydrolysis and the
conformational change in the chaperone occur simultaneously, according
to Reaction 1.
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(Reaction 1) |
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(Reaction 2) |
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(Eq. 2) |
On the basis of this work, we conclude that the mechanistic details of how the bacterial Hsp70 and the eukaryotic cytosolic Hsc70 couple ATP hydrolysis to structural transitions are different. Single turnover experiments on bovine Hsc70 have revealed the same two-step mechanism as shown in Reaction 2 (14); however, the rate-limiting step for this eukaryotic cytosolic Hsc70 is not the first step, ATP hydrolysis, it is the second step, product release. This mechanistic difference is probably related in some way to the fact that the bacterial Hsp70 chaperone DnaK depends on the cochaperones DnaJ and GrpE to accelerate ATP hydrolysis and ADP release, respectively, whereas the eukaryotic cytosolic Hsc70 chaperone is GrpE-independent (26, 27).
Although the cross-linking results in this report were interpreted in
terms of Model 1, there are two reasons why Model 2 should not be
discarded. First, a genetically engineered COOH-terminal fragment of
DnaK, with a peptide in the binding site, crystallizes in two different
forms (8). In one crystal form the -helical lid blocks the deep
peptide binding channel, while in the other crystal form the
-helical lid is rotated away from the channel, making the channel
more accessible. Possibly, these two different crystals represent the
two different E state isomers. Second, hexokinase, which has
an ATP and glucose binding core that is identical in tertiary structure
to the ATP-binding core of 70-kDa molecular chaperones (4),
equilibrates between closed and open forms in the absence of glucose
(28, 29). Given the similar tertiary structures of hexokinase and the
70-kDa chaperones, it is reasonable to postulate that the ATPase domain
of 70-kDa chaperones also equilibrates between closed and open forms in
the absence of substrates, as indicated in Model 2. Clearly, more
experiments are required before the presence of the two monomers bands
can be definitively assigned to a specific model.
Last, we address one other interpretation of the gel results. Suppose that the high affinity species, which migrates as the apparent molecular mass 90-kDa species after cross-linking, and the low affinity species, which migrates as the 70-kDa species after cross-linking, are in equilibrium in the absence of ATP, the equilibrium lies to the low affinity state, and ATP binding shifts the equilibrium completely to the low affinity state. Specifically, suppose that 30% of the DnaK molecules populate the high affinity state and 70% of the DnaK molecules populate the low affinity state in the absence of ATP; cross-linking such a sample would yield a doublet, exactly as shown in Fig. 2. On the other hand, because ATP binding shifts the equilibrium to the low affinity state, cross-linking would yield a singlet, exactly as shown in Fig. 2. In this interpretation of the gel data, polypeptide binding should shift the population of molecules from the low affinity to the high affinity state. But we have been unable to cause such a shift by incubating DnaK with a large excess of peptide and then cross-linking. In addition, the idea that DnaK predominantly populates the low affinity state in the absence of ATP also contradicts results from structural (12, 13) and kinetic (15) studies, which have shown that in the absence of ATP 70-kDa chaperone molecules populate the high affinity state. We believe the best explanation of the gel results is Model 1, where in the absence of ATP all DnaK molecules populate the high affinity state, which happens to cross-link heterogeneously. Whichever model turns out to be correct, the low affinity to high affinity structural transition in DnaK molecules occurs at exactly the same rate as DnaK-catalyzed ATP hydrolysis, consistent with Reaction 2.
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FOOTNOTES |
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* This work was supported in part by grants from the American Cancer Society and the National Institutes of Health (to S. N. W.).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 should be addressed. Tel.: 318-675-7891;
Fax: 318-675-5180; E-mail: switt1{at}lsumc.edu.
1 The abbreviations used are: Hsp70, 70-kDa heat shock protein; AMP-PNP, adenyl-5'-yl imidodiphosphate; Hsc70, 70-kDa heat shock cognate protein; PAGE, polyacrylamide gel electrophoresis.
2 The apparent molecular mass of the slower migrating DnaK monomer was 90 kDa in the 4-12% polyacrylamide denaturing gel and 110 kDa in the 10% polyacrylamide denaturing gel. We believe that the gradient gel gives a more accurate molecular mass for the slower migrating monomer than the 10% gel; therefore the slower migrating monomer is referred to as the apparent molecular mass 90-kDa species.
3
It is theoretically possible that the slow
E** E transition in DnaK is due to the two-step
reaction
E**ATP
E-ATP
E-ADP, where
a rate-limiting conformational change precedes ATP hydrolysis.
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REFERENCES |
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