©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleotide-induced Conformational Changes in the ATPase and Substrate Binding Domains of the DnaK Chaperone Provide Evidence for Interdomain Communication (*)

Alexander Buchberger (1), Holger Theyssen (2), Hartwig Schröder (1), John S. McCarty (1), Giuseppe Virgallita (3), Philipp Milkereit (1), Jochen Reinstein (2), Bernd Bukau (1)(§)

From the (1)Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neueuheimer Feld 282, D-69120 Heidelberg, Germany, the (2)Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, D-44139 Dortmund, Germany, and (3)Hoffmann-La Roche AG, CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 -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.

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) ()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.

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.


MATERIALS AND METHODS

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) .


RESULTS

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.

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.

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.

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, -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.

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.

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).

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).


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).

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) . 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.

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.


DISCUSSION

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 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.

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) .


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.

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 ATPS 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 ATPS, however, no valid conclusion could be drawn since the ATPS 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.

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 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.

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 MgATP complex at the active site in a highly coordinated manner(48) . As a result of this very specific coordination of MgATP, 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.

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. 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.


FOOTNOTES

*
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (to B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-06221-566865; Fax: 49-06221-565892.

R. McMacken, personal communication.

H. Schröder, J. Gamer, A. Buchberger, R. McMacken, and B. Bukau, manuscript in preparation.

H. Theyssen, B. Bukau, and J. Reinstein, manuscript in preparation.

The abbbreviations used are: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl glycine; CAPS, 3-(cyclohexylamino)propanesulfonic acid; mAb, monoclonal antibody; ATPS, adenosine 5`-O-(thiotriphosphate); AMP-PNP, 5`-adenylyl-,-imidodiphosphate; mAb, monoclonal antibody.

H. Schröder and B. Bukau, unpublished results.

H. Theyssen and J. Reinstein, unpublished results.

J. S. McCarty, unpublished results.


ACKNOWLEDGEMENTS

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.


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