From the Biochemisches Institut, Universität
Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland
and the § F. Hoffmann-La Roche Ltd., Pharmaceutical
Research-Infectious Diseases, CH-4070 Basel, Switzerland
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ABSTRACT |
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The polypeptide binding and release cycle of the
molecular chaperone DnaK (Hsp70) of Escherichia coli is
regulated by the two co-chaperones DnaJ and GrpE. Here, we show that
the DnaJ-triggered conversion of DnaK·ATP (T state) to
DnaK·ADP·Pi (R state), as monitored by intrinsic
protein fluorescence, is monophasic and occurs simultaneously with ATP
hydrolysis. This is in contrast with the TR conversion in the
absence of DnaJ which is biphasic, the first phase occurring
simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster,
H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates
ATP hydrolysis but also couples it with conformational changes of DnaK.
In the absence of GrpE, DnaJ forms a tight ternary complex with
peptide·DnaK·ADP·Pi (Kd = 0.14 µM). However, by monitoring complex formation between
DnaK (1 µM) and a fluorophore-labeled peptide in the
presence of ATP (1 mM), DnaJ (1 µM), and
varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3
µM), substoichiometric concentrations of GrpE were found
to shift the equilibrium from the slowly binding and releasing,
high-affinity R state of DnaK completely to the fast binding and
releasing, low-affinity T state and thus to prevent the formation of a
long lived ternary
DnaJ· substrate·DnaK·ADP·Pi complex. Under
in vivo conditions with an estimated chaperone ratio of
DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the
chaperone cycle by transient interactions with DnaK.
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INTRODUCTION |
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Heat shock proteins of the Hsp70 family are involved in essential cellular processes such as protein folding (1), the translocation of proteins through biological membranes (2), the oligomeric assembly of proteins (3) and their degradation (4). Hsp70s exert their function in conjunction with cycles of binding and hydrolysis of ATP which for their part are regulated by proteinaceous cofactors.
DnaK, the Hsp70 homolog of Escherichia coli, is controlled
by the two co-chaperones DnaJ (41 kDa) and GrpE (22 kDa), which if
acting together increase the weak ATPase activity of DnaK by 2 orders
of magnitude (5-7). DnaJ accelerates the rate of -phosphate cleveage of DnaK-bound ATP (5, 6), whereas GrpE promotes the release of
ADP (5). Several model cycles for the DnaK/DnaJ/GrpE molecular
chaperone machinery and their interaction with target polypeptides have
been proposed. In two of those models (8, 9), DnaJ rather than DnaK
interacts first with the substrate polypeptide and targets it for DnaK.
Following DnaJ-induced hydrolysis of DnaK·ATP to
DnaK· ADP·Pi, a ternary
DnaJ·substrate·DnaK·ADP·Pi complex is formed. The
ternary complex is thought to be responsible for the chaperone effect
by sequestering the substrate protein and thereby preventing it from
aggregation (8). This mechanism requires equimolar concentrations of
DnaK and DnaJ for every turn of the chaperone cycle. In another model
(6), the role of DnaJ is to convert the low-affinity DnaK·ATP form to
the high-affinity DnaK·ADP·Pi form, thereby locking the
chaperone onto the target polypeptide. In this model, DnaJ may act in a
catalytic manner and fulfill its action without forming a stable
ternary complex with peptide·DnaK·ATP. Such a catalytic effect of
DnaJ has been directly shown in the binding of the
32
heat shock transcription factor to DnaK; DnaJ promotes the binding without becoming itself part of the DnaK·
32 complex
(10).
By measuring the kinetics of the individual steps of the DnaK/DnaJ/GrpE
chaperone cycle, we have recently shown that at intracellular concentrations of chaperone and co-chaperones, the
rate-limiting step of the cycle is the DnaJ-triggered conversion of the
ATP form of DnaK (T state) with low affinity for target peptides to its
ADP form (R state) with high affinity for target peptides. The ratio of
the rates of the TR and R
T conversions indicates, that the R
state is only a minor form of DnaK (7). Since DnaK in the presence of
ATP and the absence of the co-chaperones has no or only a minor
chaperone effect (11) and since the peptide segment bound to the R
state of DnaK is in an extended conformation (12) and thus represents
the result of any disaggregating and disentangling action, the power
stroke, i.e. the step which underlies the chaperone effect
of DnaK, must be the slow ATP-driven T
R conversion. ATP hydrolysis
might provide energy for performing conformational work on the peptide
during the T
R conversion. In this hypothetical chaperone cycle both
the ternary DnaJ·polypeptide· DnaK·ADP·Pi
complex and the polypeptide·DnaK·ADP·Pi complex
are only short-lived intermediates that are not directly responsible for the chaperone effect (7). The present study investigates the
dependence of the chaperone cycle on the concentrations of the
co-chaperones. The results support the notion that substoichiometric amounts of DnaJ and GrpE suffice to control the DnaK chaperone cycle.
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MATERIALS AND METHODS |
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Proteins--
DnaK was purified and prepared for experimentation
as described (7, 13). Protein concentration was determined
photometrically with a molar absorption coefficient of
280 = 14500 M
1
cm
1 (14). Purified DnaK contained < 0.1 mol of ADP
per mol of DnaK (15). The concentrations of DnaK stock solutions were
in the range of 60 to 115 µM. All concentrations of DnaK
and the co-chaperones refer to their protomers. GrpE and DnaJ were
prepared as described previously (16, 17). Their concentrations were
determined by quantitative amino acid analysis. The stock solution of
DnaJ was 120 µM in 50 mM Tris chloride, pH
7.7, 100 mM NaCl, and that of GrpE was 240 µM
in 50 mM Tris chloride, pH 7.7. The preparations of DnaK
and both co-chaperones were more than 95% pure as estimated by
SDS-polyacrylamide gel electrophoresis. All three proteins were stored
at
80 °C.
Labeled Peptide-- Peptide p5 (CLLLSAPRR) was labeled with the fluorescent reporter group 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)1 (Molecular Probes, Eugene, OR) at the sulfhydryl group of the NH2-terminal cysteine residue to give a-p5 and purified as described (7). The concentrations of the stock solutions of a-p5 were 24-34 µM.
Buffers-- All experiments were performed in assay buffer (25 mM Hepes-NaOH, 100 mM KCl, pH 7.0), if not indicated otherwise, at 25 °C.
Fluorescence Spectra-- Fluorescence spectra of the acrylodan-labeled peptide were recorded with a Spex Fluorolog spectrofluorimeter in a 0.2-cm cuvette using excitation slits of 4.6/4.6 nm and emission slits of 18.5/9.2 nm. The excitation wavelength was set at 370 nm. Spectra of the intrinsic fluorescence of DnaK were either recorded with the SPEX spectrofluorimeter with excitation slits of 0.9/0.9 nm and emission slits of 18.5/9.2 nm (experiment of Fig. 1) or a Perkin-Elmer LS 50B spectrofluorimeter in a 1-cm cuvette using excitation and emission slits of 5 nm (experiment of Fig. 2). Excitation wavelength was set in both cases at 290 nm.
Kinetic Measurements-- For measuring changes in the fluorescence of acrylodan-labeled peptides, emission was recorded at 500 nm; for following changes in the intrinsic fluorescence of DnaK, emission was recorded at 340 nm. Single-turnover experiments were measured with the SPEX fluorimeter with excitation slits of 0.9/0.9 nm and emission slits of 18.5/9.2 nm (experiment of Fig. 3B) or the Perkin-Elmer spectrofluorimeter, with excitation and emission slits of both 5 nm (experiment of Fig. 3A) using a 0.4-cm cuvette with a magnetic stirrer. To ensure rapid mixing, the reactant by which the reaction was started was injected with a syringe. The dead time was thus reduced to approximately 2 s. The experiments of Figs. 6-8 were performed with a SF-61 stopped-flow spectrofluorimeter (Hi-Tech Scientific, Salisbury, UK) with a dead time of 1 ms. For measuring the fluorescence of labeled target peptides, the excitation wavelength was set at 370 nm and the emission light passed through a GG455 cut-off filter. The kinetic data obtained with the spectrofluorimeters were fitted with the program Sigma Plot (Jandel Scientific), which provides the asymptotic standard deviation of the calculated parameters for each evaluation. In the case of the stopped-flow experiments, the average of 3-10 measurements was fitted to a single or double exponential decay or rise function.
Single-turnover ATPase Assay--
To preform DnaK·ATP
complexes, DnaK was incubated with [2,5',8-3H]ATP (*ATP;
Amersham) for 10 min at 25 °C. The complex was separated from excess
*ATP by rapid size-exclusion chromatography with NICK columns
(Pharmacia Biotech Inc.) at 4 °C in assay buffer containing 5 mM MgCl2 according to McCarty et al.
(6). To determine the rate of -phosphate cleavage, 50 µl of
complex were incubated for 5 min at 25 °C, and at the indicated
times, the reaction was stopped by mixing 5-µl samples with 5 µl of
26 M formic acid. Samples were spotted onto
poly(ethyleneimine)-cellulose thin-layer plates, and *ADP was separated
from *ATP in 1 M HCOOH/0.7 M LiCl. *ATP and
*ADP were quantified by liquid-scintillation counting (5).
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RESULTS AND DISCUSSION |
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By monitoring changes in the tryptic digestion pattern, three different conformational states of DnaK can be detected, the nucleotide-free state of DnaK and the ADP- and ATP-liganded states (18). Another possibility to discern the different conformational states of DnaK is by following the intrinsic tryptophan fluorescence of DnaK upon addition of nucleotides. DnaK contains a single tryptophan residue (Trp-102) in its ATPase domain. By this technique, Theyssen et al. (19) recently confirmed the existence of three different conformational states of DnaK. We obtained virtually the same results, which among others are reproduced in Fig. 1 to facilitate understanding of the experiments below. While addition of ATP to nucleotide-free DnaK (Fig. 1, spectrum a) caused a blue shift from 349 to 345 nm and led to a 18% decrease of the relative fluorescence at 349 nm (spectrum b), hydrolysis of DnaK-bound ATP to ADP and Pi reversed the blue shift and increased the fluorescence again to a value 4% higher than that of the nucleotide-free state (spectrum c). In this experiment, a slight excess of ATP over DnaK was used (DnaK, 1 µM; ATP, 1.4 µM).
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The DnaJ-induced Change in Intrinsic Fluorescence of DnaK·ATP Is Due to Hydrolysis of ATP and Not to DnaK·DnaJ Complex Formation-- Since both DnaJ and GrpE are devoid of tryptophan (20, 21), effects of the co-chaperones on the tryptophan fluorescence of DnaK can easily be observed. Addition of DnaJ to DnaK in the presence of excess ATP (1 mM) increased the fluorescence at 349 nm from 82% of that of the nucleotide-free state to 90% and reversed the ATP-induced blue shift, resulting in a fluorescence spectrum (Fig. 1, spectrum d) intermediary to the spectra of nucleotide-free and ATP-liganded DnaK (spectra a and b). In a recently published report suggesting a model of the DnaK/DnaJ/GrpE reaction cycle, DnaJ was also found to increase the fluorescence of DnaK in the presence of ATP. The increase was interpreted as being due to formation of a DnaK·DnaJ complex (22). To further characterize the mode of interaction between DnaK and DnaJ in the presence of ATP, we titrated DnaK with increasing concentrations of DnaJ in the presence of 1 mM ATP (Fig. 2). The increase in fluorescence followed an approximately hyperbolic saturation curve and reached a maximum at about 2 µM DnaJ. On first sight, this increase in fluorescence might indeed be attributed to formation of a DnaK·DnaJ complex. However, another possible explanation is that the increase in fluorescence upon addition of DnaJ is due to an increase in the steady-state concentration of DnaK·ADP·Pi. Raising the concentration of DnaJ increases the rate of DnaJ-stimulated hydrolysis of DnaK·ATP to DnaK·ADP·Pi2, whereas the rate of exchange of DnaK-liganded ADP and Pi against ATP remains unchanged. To test this interpretation, we measured the DnaJ-induced spectral alterations of DnaK at different concentrations of ATP (1 mM, 36 µM, and 1.4 µM; spectra d, e, and f, respectively, in Fig. 1). Lowering the concentration of ATP was expected to slow down ADP/ATP exchange and thus to increase the steady-state concentration of DnaK·ADP·Pi. The increase in fluorescence upon addition of DnaJ indeed depended inversely on the concentration of ATP and reached a maximum under single-turnover conditions when approximately equimolar concentrations of DnaK and ATP prevented ADP/ATP exchange. Under single-turnover conditions, DnaK adopted the same conformation as DnaK which was allowed to hydrolyze bound ATP completely to ADP·Pi in the absence of DnaJ (Fig. 1, spectra f and c).
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The DnaJ-triggered Conformational Change TR of DnaK Occurs
Concomitantly with ATP Hydrolysis--
Recently, the change in
intrinsic fluorescence accompanying the spontaneous conversion of
DnaK·ATP to DnaK·ADP·Pi in the absence of DnaJ was
shown to be a two-step process with k1 = 0.0018 s
1 and k2 = 0.007 s
1
(19). Since the rate k1 was virtually identical
to the rate of ATP hydrolysis (khyd = 0.0015 s
1), the first step was ascribed to the hydrolysis of ATP
and the second step to a subsequent structural change. We have shown
previously that the T
R conversion of DnaK (1 µM),
triggered by DnaJ (1 µM) and measured under
single-turnover conditions, is a monophasic process with
kobs = 0.044 s
1 (7). The
tryptophan spectrum of the isolated ATPase domain (DnaK1-385) shows
only minor changes upon binding of ATP and does not undergo the
characteristic ATP-induced blue shift. Therefore, it was concluded that
the ATP-induced changes in intrinsic fluorescence of the complete DnaK
molecule derive from interaction of the exposed Trp residue (Trp-102)
in the ATPase domain with the peptide-binding domain (18). According to
this notion, the DnaJ-triggered changes in fluorescence of DnaK reflect
the T
R conversion of DnaK. Because the affinity for peptide ligands
is low in T-state DnaK and is enhanced in the R state, the slow T
R
transition ought to be observable also by following the binding of a
target peptide. We followed the T
R transition under single-turnover
conditions by intrinsic fluorescence and compared its rate with that of
complex formation between DnaK and the fluorescence-labeled peptide
a-p5 in the course of the T
R conversion (Fig.
3A). The nonapeptide p5 (for its sequence, see "Materials and Methods") corresponds to the main
binding site for DnaK of the 23-residue prepiece of mitochondrial aspartate aminotransferase3;
a-p5 is a high-affinity ligand for DnaK with Kd
values of 5.1 µM and 0.07 µM in the
presence of ATP and ADP plus Pi, respectively (7). The
change in intrinsic fluorescence and the binding of the peptide
proceeded with virtually the same rate of 0.003 s
1. Under
the conditions used, 16 and 93% of a-p5 are expected to be bound to T-
and R-state DnaK, respectively. Indeed, during the T
R transition,
the overall reaction amplitude was 5.3-fold expanded as compared with
the amplitude for the reaction a-p5 + DnaK·ATP before the addition of
DnaJ.
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Stimulation of the Rate of -Phosphate Cleavage Is Due to
Formation of a Transient DnaK·DnaJ Complex--
A low
concentration of DnaJ (DnaK, 1.2 µM; DnaJ, 22 nM) was reported previously to shorten the half-life of
DnaK-bound ATP from 9.1 to 1.8 min, indicating that DnaJ stimulates the
ATPase activity of DnaK in a catalytic manner (10). We measured the DnaJ-induced acceleration of the single-turnover rate of ATP hydrolysis as a function of the concentration of DnaJ (Fig.
4). The stimulation depended linearly on
the concentration of DnaJ. At concentrations of DnaK and DnaJ of 1 and 0.1 µM, respectively, DnaJ stimulated the ATPase
approximately 20-fold. This degree of acceleration is of physiological
relevance since the concentrations of DnaK and DnaJ in E. coli are in the micromolar range (20, 24), and their ratio is
approximately 10:1 (20). At a concentration of DnaJ of 2 µM, a >900-fold acceleration was reached. The plot of
the rate of single-turnover ATP hydrolysis as a function of the
concentration of DnaJ indicates the Kd value for DnaJ in the DnaK·ATP·DnaJ complex to be considerably higher than 2 µM (Fig. 4B), which agrees with the notion of
a relatively weak DnaK·ATP-DnaJ interaction.
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In the Absence of GrpE, DnaJ Forms a Ternary Complex with Peptide·DnaK·ADP·Pi-- DnaJ is known to form ternary complexes with peptide·DnaK in the presence of ATP (8, 9, 25, 26). To examine whether DnaJ also forms a ternary complex with peptide a-p5 and DnaK in the presence of ADP and Pi, a-p5·DnaK·ADP·Pi complex was preformed under conditions such that all peptide was bound. Addition of DnaJ to the preformed complex indeed led to a further increase in fluorescence in the region from 440 to 500 nm and was accompanied by a blue shift from 500 to 484 nm, indicating an additional interaction between the label of DnaK-bound peptide and DnaJ (Fig. 5). From the titration of a-p5·DnaK·ADP·Pi with DnaJ, a Kd value of 0.14 µM was calculated.
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Substoichiometric Concentrations of GrpE Prevent Formation of the Ternary DnaJ·Peptide·DnaK·ADP·Pi-Complex-- At equimolar concentrations of DnaK and the two co-chaperones, GrpE reverses the DnaJ-induced conformational switch of DnaK from the T to the R state, indicating that the GrpE-mediated ADP/ATP exchange is faster than the DnaJ-triggered hydrolysis of DnaK-bound ATP (7). To titrate this effect of GrpE, the formation of the a-p5·DnaK complex in the presence of ATP was followed at constant concentrations of DnaK and DnaJ (1 µM each) and varying concentrations of GrpE (Fig. 7A). The rate kobs, in the case of a single-step process, or kobs1, in the case of a double-step process, and the total reaction amplitudes were determined. The rate for the first phase increased at least 110-fold at 0.5 µM GrpE compared with the reaction without GrpE (Fig. 7B). Apparently, catalytic concentrations of GrpE maintain DnaK in its fast-binding-and-releasing T state. A further increase in the concentration of GrpE decelerated the binding reaction.
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Conclusion-- The aim of this study was to gain more detailed insight into the regulation of the DnaK chaperone cycle by the co-chaperones DnaJ and GrpE. In particular, the question was addressed whether the co-chaperones participate in a stoichiometric manner in the chaperone cycle as proposed in several hypothetical models or whether they rather serve as regulatory catalysts.
In several studies, ternary DnaJ·polypeptide·DnaK complexes have been isolated. In the presence of ATP, denatured luciferase coelutes with DnaK and DnaJ in a higher molecular mass complex of ![]() |
FOOTNOTES |
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* This work was supported in part by the Swiss National Science Foundation Grant 31.36542.92, the EMDO-Stiftung, Zürich, the Fonds für Medizinische Forschung der Universität Zürich, and the Ernst-Göhner Stiftung, Zürich.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.: 41-1-635-55-60; Fax: 41-1-635-68-05; E-mail: christen{at}biocfebs.unizh.ch.
1 The abbreviations used are: acrylodan, 6-acryloyl-2-dimethylaminonaphthalene; *ATP, [2,5',8-3H]ATP.
2 ADP and Pi bind to DnaK with Kd values of 0.13 and 0.45 µM, respectively (19). They are thus assumed to be exchanged together against ATP.
3 S. Gisler, E. V. Pierpaoli, and P. Christen, manuscript in preparation.
4 E. V. Pierpaoli, E. Sandmeier, and P. Christen, unpublished data.
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REFERENCES |
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