©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Divergent Effects of ATP on the Binding of the DnaK and DnaJ Chaperones to Each Other, or to Their Various Native and Denatured Protein Substrates (*)

(Received for publication, February 10, 1995; and in revised form, June 6, 1995)

Alicja Wawrzynów (§) Maciej Zylicz

From the Division of Biophysics, Department of Molecular Biology, University of Gdansk, 24 Kladki, Gdansk 80-822, Poland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Using the native proteins P, O, , and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses the ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.


INTRODUCTION

The highly conserved and ubiquitous 70-kDa heat shock proteins (Hsp70), or the prokaryotic DnaK homologues, function as molecular chaperones in a variety of cellular processes (reviewed by Gething and Sambrook(1992), Hendrick and Hartl(1993), Georgopoulos et al.(1994), and Craig et al.(1994)). For example, Hsp70 chaperones have been shown to be involved in the binding of nascent polypeptides (Beckman et al., 1990), protein transport (Neupert et al., 1990; Scherer et al., 1990; Wild et al., 1992; Hendrick et al., 1993; Brodsky et al., 1993), the disassembly of clathrin-coated vesicles (Chappell et al., 1986; Schmid and Rothman, 1985a, 1985b; Schmid et al., 1985; Prasad et al., 1994), the activation of the p53 anti-oncogene to bind to specific DNA sequences (Hupp et al., 1992), the DNA replication of bacteriophages (Zylicz, 1993) and P1 (Wickner et al., 1992), and the autoregulation of the heat shock response (Yura et al., 1993), as well as in vivo proteolysis (Sherman and Goldberg, 1992). The Hsp70 chaperones can function in these diverse cellular processes because they exhibit promiscuous substrate binding properties (BlondElguindi et al., 1993a; Gragerov et al., 1994). For instance, Hsp70 generally recognizes nascent polypeptides chains, peptides in the extended conformation (Flynn et al., 1991; Landry et al., 1992), and unfolded proteins (bovine pancreatic trypsin inhibitor; RNA polymerase, CMLA, (^1)rhodanese; for a review, see Hendrick and Hartl(1993)). Through binding to the hydrophobic unfolded regions of proteins, the Hsp70 chaperones not only prevent protein aggregation, but can even disassociate protein aggregates, thus facilitate protein folding/assembly (Pelham, 1986; Skowyra et al., 1990; Schroder et al., 1993; Ziemienowicz et al., 1993). (^2)In addition, Hsp70 can bind and activate native proteins, which necessitate conformational changes for their activation, i.e. P, O (Liberek et al., 1990), RepA (this paper), p53 (Hupp et al., 1992), and (Liberek et al., 1992; Gamer et al., 1992; Liberek and Georgopoulos, 1993).

Crucial to Hsp70's mode of action is its ability to bind and hydrolyze ATP (Zylicz et al., 1983; Welch and Feramisco, 1985). Hsp70 has a relatively high affinity for ATP and an extremely weak ATPase activity (for a detailed review see, McKay et al.(1994) and Hightower et al.(1994)), which can be stimulated up to 20-fold by peptides or certain protein substrates (Hightower et al., 1994; Blond-Elguindi et al., 1993b; Buchberger et al., 1994). The binding and hydrolysis of ATP results in conformational changes to the various Hsp70 family members (Liberek et al., 1991b; Palleros et al., 1992; Banecki et al., 1992). Recently it was suggested (under conditions where DnaJ was not present) that the binding, but not ATP hydrolysis, is required for the disassociation of Hsp70 from its substrate (Palleros et al., 1993; Schmid et al., 1994).

Most models addressing the question of how Hsp70 functions in protein transport or folding assume that Hsp70 bound to ADP (Hsp70-ADP) is the form exhibiting the highest affinity toward the substrate and ATP hydrolysis is required only for the regeneration of such a form (for review, see Craig et al.(1994)). This conclusion was based on the findings that Hsp70 family members form a stable complex with various substrates in the presence of ADP but not ATP (Palleros et al., 1991). However, recently stopped-flow measurements have revealed that the DnaK chaperone exhibits a faster on rate for peptides when bound to ATP than ADP, but it exhibits an even faster off rate when bound to ATP. Thus, overall the DnaK-substrate complex is less stable in the presence of ATP than ADP (Schmid et al., 1994).

In this paper we show that the product of ATP hydrolysis, namely the DnaK-ADP form possesses limited affinity to the protein substrates but binds efficiently to the DnaJ protein.


MATERIALS AND METHODS

Purified Proteins

The Escherichia coli and bacteriophage replication proteins O, P, DnaK, GrpE, and DnaJ were purified as described previously by Zylicz et al. (1989). The replication activity of these proteins was tested as described by Zylicz et al.(1985). The transcription factor was purified and its transcription activity estimated as described by Liberek et al.(1992). The P1 replication protein, RepA, was a kind gift of Dr. Dhruba K. Chattoraj (NIH). alpha-Carboxylmethylated lactalbumin (CMLA) was purchased from Sigma. The purity of all proteins was >95%. The DnaKc94 mutant protein which possesses deletion of 94 amino acids from the C-terminal domain of the DnaK protein was a kind gift of Dr. Anna Maddock (University of Utah).

Anti-DnaK and anti-DnaJ rabbit sera were generated as described by Zylicz and Georgopoulos(1984). Protein concentrations were estimated by the Bio-Rad assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie Blue staining.

Enzyme-linked Immunosorbent Assay (ELISA)

The ELISA used for detection protein-protein interactions was a modified version of that published by Wawrzynow et al. (1995a). The indicated proteins (0.5 µg/well) were added to a 96-well microtiter plate in 50 µl of PBS buffer. In control experiments we showed that 0.5 µg of protein per well is enough to allow saturation of the amount protein bound to the well. After a 1-h incubation at room temperature, the solution was removed and the wells were washed 4 times with PBS containing 0.2% bovine serum albumin (PBS/BSA, 100 µl). The first three washes were immediate, whereas the last wash solution was incubated for 1 h before removal. The wells were then washed with buffer A (25 mM Hepes/KOH, pH 7.6, 150 mM KCl, 25 mM NaCl, 10 mM MgCl(2), 0.1 mM EDTA, 2 mM dithiothreitol, 5%(v/v) glycerol, and 0.05% Triton X-100) supplemented with 0.2% BSA (buffer A/BSA) and the indicated protein(s) were added in 50-µl aliquots in buffer A/BSA. The reaction mixture was supplemented with nucleotides as indicated (1 mM), and the volume of the reaction was adjusted to 60 µl. The 0.05% Triton X-100 was present during binding assay to prevent self-aggregation of DnaK and DnaJ chaperones. After a 30-min incubation at room temperature, glutaraldehyde was added to a final concentration of 0.1% and incubation was allowed to proceed for an additional 5 min at room temperature. Presented in this paper the experiments were repeated without cross-linking with glutaraldehyde and the same essential results were obtained. The wells were then washed with buffer A/BSA followed by three washes of PBS/BSA. The appropriate antibody was then added (1/20,000 dilution for anti-DnaK serum, 1/10,000 dilution for anti-DnaJ serum) in buffer A/BSA (100 µl) and incubated for 2 h at room temperature. Unbound protein was removed and the wells were washed 4 times with PBS/BSA. Following this, the secondary antibody was added in PBS/BSA (100 µl) and incubated for 45 min. Following protein removal and four washes as above, colorimetric detection was done with the Bio-Rad TMB peroxidase EIA substrate kit. The reaction was stopped with an equal volume (100 µl) of 4 N sulfuric acid. Absorbance (490 nm) was measured with a Molecular Devices TermoMax microplate reader. Each experiment was repeated 6 times and the average value was estimated. In most cases the standard deviation was less than 5%.

Size Exclusion Chromatography

Chaperone proteins, along with the appropriate substrates at the concentrations indicated in the various figure legends (50-µl reaction mixture) were incubated for 30 min at 30 °C in buffer B (same as buffer A except no Triton X-100 was used). The mixture was then injected onto a size exclusion chromatography Superdex 200 (Pharmacia Biotech Inc.) column equilibrated with buffer B. Where stated in the figure legends, either the buffer in the reaction mixture or during equilibration, the column may also have contained 200 µM of the appropriate nucleotide. Chromatography was carried out at a flow rate 0.3 ml/min at room temperature using a Gold HPLC system (Beckman) equipped with a diode array detector.


RESULTS

Binding of the DnaK Chaperone to Various Protein Substrates

The affinity of DnaK for various protein substrates was tested using two techniques, a sensitive ELISA and size exclusion HPLC. As model substrates, we decided to use four native protein substrates namely O, P, RepA, and , all four known previously to interact with chaperone proteins both in vivo and in vitro (for a review, see Georgopoulos et al.(1994)). All four of these proteins were in their native state and biochemically active in an in vitro or P1 DNA replication assay (in the case of O, P, and RepA) and a transcription assay (in the case of ) as described previously (Zylicz et al., 1989; Wickner et al., 1992; Liberek et al., 1992). In addition to these native proteins, an unfolded protein in the extended configuration was chosen, CMLA. CMLA is thought to mimic a nascent polypeptide substrate emerging from a ribosome or traversing a biological membrane (Langer et al., 1992; Palleros et al., 1993; Cyr et al., 1992, 1994). As shown in Fig. 1, DnaK exhibits the highest affinity to O, P, and RepA. It was previously reported that both O and P interact with DnaK (Liberek et al., 1990). A DnaK-RepA complex in the absence of DnaJ was not previously detected by others (Wickner et al., 1992). The CMLA and proteins bind less efficiently to the DnaK chaperone (Fig. 1A). Theoretically it is possible that the conformation of the substrate bound to the ELISA plate is different from the conformation of the substrate in the solution. Also the mode of binding to the plates could be different among different proteins, affecting the sites available for interaction with chaperones. To eliminate these possibilities we bound CMLA (the similar result was obtained with P; result not shown) to the ELISA plate and measured the affinity of DnaK preincubated with various concentrations of P, O, RepA, or CMLA. It means that all these protein substrates were incubated with DnaK under their optimal activity conditions. As shown in Fig. 1B, again O, P, and RepA compete more efficiently with CMLA for binding to DnaK. Both bound to the ELISA plate and when free in a solution, has been shown to have a low affinity to DnaK chaperone. This is consistent with our previous findings where the binding of to DnaK was monitored using the glycerol gradient centrifugation (Liberek et al., 1992). Moreover, the experiment in which bound to the ELISA plate specifically interacts with the core of RNA polymerase and competes for this binding, additionally confirms that the conformation of the protein on the plate and in the solution is at least similar. The P protein bound to the ELISA plate specifically interacts with the DnaB helicase and O bound to the ELISA plate is efficiently recognized by the ClpX chaperone (Wawrzynow et al., 1995a). (^3)As described in this paper, the ELISA for protein-protein interaction is also sensitive to the tertiary structure of some substrates. The heat inactivated luciferase bound to the ELISA plate is more efficiently recognized by ClpX or DnaK chaperones than the native form of luciferase (Wawrzynow et al., 1995a). (^4)In control experiments we also tested the influence of reducing conditions on the affinity of DnaK to bind to CMLA. When CMLA (327 mM stock solution in 10 mM HCl, pH 2) was diluted in a binding buffer containing 5 or 10 mM dithiothreitol, DnaK exhibited only a slightly higher (5-10%) affinity toward CMLA than when only 2 mM dithiothreitol was present in the binding buffer (Fig. 1A).


Figure 1: Interaction of the DnaK and DnaJ molecular chaperones with various protein substrates. Panels A and C, the various protein substrates, namely: , RepA; bullet, P; , O; ▴, ; Delta, CMLA; and ▪, BSA, were preincubated (0.5 µg/well) in ELISA plate wells in PBS buffer as described under ``Materials and Methods.'' Following the washing procedure with PBS/BSA and A/BSA buffers, an increasing amount of either DnaK (panel A) or DnaJ (panel C) in buffer A/BSA was added and the volume adjusted to 60 µl. After a 30-min incubation at room temperature, the proteins were cross-linked with glutaraldehyde as described under ``Materials and Methods.'' The amount of DnaK (panel A) or DnaJ (panel C) bound to the various protein substrates was detected using either anti-DnaK or anti-DnaJ serum, respectively, as described under ``Materials and Methods.'' Panels B and D, the CMLA (0.5 µg/well) was preincubated on ELISA plate, as described above. After washing with PBS/BSA and A/BSA buffer, increasing amounts of protein substrates: , RepA; bullet, P;. , O; ▴, ; and , CMLA were preincubated for 15 min at 30 °C with 80 µg of DnaK (panel B) and 40 µg of DnaJ (panel D) in A/BSA buffer was added and incubation proceeded for an additional 15 min. After cross-linking with glutaraldehyde and washing, the amount of DnaK (panels A and B) and DnaJ (panels C and D) bound to CMLA was detected using either anti-DnaK or anti-DnaJ serum, respectively.



Previously we had shown that DnaK possesses a weak ATPase activity resulting in the hydrolysis of 1 molecule of ATP every 10 min (Zylicz et al., 1983; Liberek et al., 1991a). In addition, it was shown that ATP causes the release of DnaK from P, O, unfolded bovine pancreatic trypsin inhibitor, and RepA, albeit inefficiently (Fig. 2) (Liberek et al., 1990, 1991b). In the case of and CMLA, which possess a lower (without nucleotides) affinity toward DnaK, the ATP-dependent dissociation of DnaK from these substrates was more pronounced (Fig. 2). These results suggest that the presence of ATP simply shifts the equilibrium of the DnaK/substrate reaction in favor of dissociation. Under these conditions, ADP has only a modest effect on the binding of DnaK to some of its protein substrates (Fig. 2). Surprisingly, the nonhydrolyzable ATP analogue (AMP-PCP) efficiently stimulates the binding of all protein substrates to DnaK (Fig. 2). The stimulatory effect of AMP-PCP was even more pronounced for the and CMLA substrates (Fig. 2). In fact, in the presence of AMP-PCP, DnaK exhibits a similar binding property for all tested protein substrates (Fig. 2). Recently, Palleros et al.(1993) reported that the ATPS nonhydrolyzable analogue causes the partial release of CMLA from its complex with DnaK. To address this apparent discrepancy we tested two other nonhydrolyzable ATP analogues for their effect on substrate binding to DnaK. All three ATP analogues tested, namely, AMP-PCP, AMP-PNP, and ATPS, stimulated binding of DnaK to P or CMLA to the same extend (Fig. 3). Nevertheless, because during the course of these experiments we found that ATPS was unstable we decided to exclusively use AMP-PCP.


Figure 2: Nucleotides modulate the binding of the DnaK chaperone to its various substrates. The various protein substrates (indicated in left top corner of each panel) were preincubated (0.5 µg/well) in ELISA plate wells as described under ``Materials and Methods.'' After the blocking and washing procedures, an increasing amount of DnaK chaperone was added in buffer A/BSA supplemented with the indicated nucleotide (1 mM): bullet, ATP; , ADP; , AMP-PCP; and , without nucleotide. After incubation for 30 min at room temperature and cross-linking with glutaraldehyde, the amount of the DnaK chaperone bound to various protein substrates was estimated using an anti-DnaK serum as described under ``Materials and Methods.''




Figure 3: Effect of nonhydrolyzable ATP analogues on the binding of the DnaK chaperone to either P or CMLA. P (0.5 µg/well, panel A) or CMLA (0.5 µg/well, panel B) proteins were bound to ELISA plate wells as described under ``Materials and Methods.'' The binding of increasing amounts of the DnaK chaperone in the presence (1 mM) of: , AMP-PCP; ▪, ATPS; ▴, AMP-PNP; or , without nucleotides, was measured as described in the legend of Fig. 2and detailed under ``Materials and Methods.''



Next, we examined the influence of nucleotides on the stability of the DnaK-substrate complexes. DnaK was preincubated with P or CMLA in the presence of AMP-PCP for 30 min at room temperature (similar results were obtained when no nucleotides were used), then AMP-PCP and unbound protein were removed. Following this, fresh buffer with the indicated nucleotides were added and the reaction was stopped at various times by cross-linking with glutaraldehyde. The DnaK-P or DnaK-CMLA complexes were relatively stable in the absence of nucleotides or in the presence of ADP but in the presence of ATP nearly all DnaK bound to either CMLA or P was released within 5 min (results not shown).

To further substantiate our results obtained from the ELISA experiments, we also performed size exclusion chromatography using a Superdex HR 200 column (Pharmacia). When P was incubated with DnaK (2:1 molar ratio, respectively) a peak was detected which corresponds with a dimer of P bound to a monomer of DnaK (Fig. 4). This protein complex was previously identified by us (Osipiuk et al., 1993). The presence of ATP in the premixture did not disrupt the DnaK-P complex (results not shown). However, when ATP was present both in the premixture and in the column buffer, the DnaK-P complex was disrupted (Fig. 4). This result suggests that when ATP is not present in the column buffer, the P-DnaK complex reforms due to the high affinity of DnaK to P. In contrast to P, the presence of ATP in the premixture only was sufficient to disrupt the DnaK-CMLA complex (Fig. 4), again suggesting that DnaK forms a weaker complex with CMLA than P (Fig. 1A). The presence of ADP had no detectable effect on the DnaK-P or DnaK-CMLA complexes (Fig. 4). In good agreement with the reported ELISA-type of experiments (Fig. 2) is the observation that the presence of AMP-PCP in the premixture stimulates binding of DnaK to P (Fig. 4). An even more pronounced stimulatory effect of AMP-PCP was found for the CMLA protein substrate than for P (Fig. 4). Interestingly, when AMP-PCP or ATPS were also present in the column buffer, a lower amount of CMLA was found in a complex with DnaK, suggesting that nonhydrolyzable ATP analogues could also destabilize the DnaK-CMLA complex (Palleros et al., 1993).^3


Figure 4: Isolation of DnaK-CMLA and DnaK-P complexes using size exclusion HPLC on Superdex 200 column. DnaK protein (35 µg) was preincubated with CMLA (15 µg, panel A) or P (26 µg, panel B) in buffer B (50 µl) for 30 min at 30 °C in the presence or absence of ATP, AMP-PCP, or ADP (1 mM) and injected onto a Superdex 200 column equilibrated with buffer B. In the case when DnaK was preincubated with P and ATP, 200 µM ATP was also present in the column buffer. Bottom HPLC profiles represent control experiments where DnaK protein or P protein alone were chromatographed under the same conditions (column buffer supplemented with 200 µM ATP). The Superdex 200 column was calibrated with the following Bio-Rad molecular weight standards: 1, thyroglobulin (670 kDa); 2, bovine -globulin (158 kDa); 3, chicken ovalbumin (44 kDa); 4, equine myoglobin (17.5 kDa).



DnaJ Exhibits a Different Spectrum of Substrate Binding Affinities Than DnaK

The affinity of DnaJ for the O, RepA, P, , and CMLA proteins was again monitored using the described ELISA. The DnaJ has the highest affinity for RepA and substrates (Fig. 1, C and D). There were no detectable differences in the binding affinities when ATP, ADP, or AMP-PCP were used (results not shown), suggesting that nucleotides do not modulate the binding properties of DnaJ. To verify these protein interactions using another method, we again performed size exclusion HPLC. Using this assay, we could only detect a complex between DnaJ and RepA (Wawrzynow et al., 1995b) and DnaJ-. (^5)Complexes between DnaJ and O, P, or CMLA were not detectable by this method (Wawrzynow et al., 1995b; results not shown), suggesting that the weak interactions detected with the ELISA were probably transient in nature, and therefore those DnaJ-substrate complexes were possible to capture only through cross-linking with glutaraldehyde.

Role of Nucleotides in the Interactions between DnaK and DnaJ

Recently we have shown that DnaJ and GrpE jointly stimulate DnaK's ATPase activity up to 50-fold, while the DnaJ alone stimulates this reaction only up to 2-3-fold (Liberek et al., 1991a). This findings suggest that DnaJ and DnaK physically interact. Using the ELISA we tested the influence of the various nucleotides on the interaction between DnaK and DnaJ. Interestingly, these nucleotides were found to influence the DnaK-DnaJ interactions in an opposite manner as compared to that of the DnaK-substrate interactions (compare Fig. 2and Fig. 5). Specifically, ATP dramatically increased DnaK's affinity toward DnaJ, while AMP-PCP weakened this interaction (Fig. 5). We also found that ADP slightly stimulated the binding of DnaK to DnaJ, compared to the situation when no nucleotide was present.^3 Therefore, we conclude that the nature of the interaction between DnaK and DnaJ is fundamentally different than that of the DnaK-substrate interaction. Moreover, the binding of DnaK to DnaJ is specific, since a single amino acid substitution in DnaJ, His-Gln (DnaJ) practically eliminates the DnaJ-DnaK interaction, but not the DnaJ-substrate interaction (Wall et al., 1995). To support the specificity of the DnaJ-DnaK complex formation we tested the interaction of DnaJ with DnaK mutant lacking 94 amino acids from its C-terminal domain. No detectable interaction between DnaJ and DnaKc94 was observed (Fig. 5).


Figure 5: Interaction of the DnaJ with DnaK or DnaKc94 in the presence of nucleotides. The DnaJ or BSA (0.5 µg/well) was loaded on the ELISA plate wells in PBS buffer and following binding, washing, and blocking procedures, as described under ``Materials and Methods,'' the increasing amounts of DnaK or DnaKc94 protein in buffer A/BSA, in either the absence or presence of ATP (1 mM) and AMP-PCP (1 mM), was added. The reaction was preincubated for 30 min at room temperature and cross-linked with glutaraldehyde as described under ``Materials and Methods.'' The amount of DnaK protein bound to DnaJ was estimated using an anti-DnaK serum as described under ``Materials and Methods.''



To verify the results obtained with ELISA we performed size exclusion HPLC. Under the described conditions the DnaK protein eluted as an asymmetric monomer (Montgomery et al., 1993).^3 The DnaJ protein behaved as a dimer, as described previously (Zylicz et al., 1985). When both proteins were mixed together, in the absence of ATP, their elution profiles were unchanged, i.e. there was no detectable interaction (Fig. 6). When ATP was added during the preincubation there was no noticeable interaction (Fig. 6). However, when ATP was present in both the premixture and in the column buffer, we did observe an additional peak, suggesting that the DnaK and DnaJ proteins interact in an ATP-dependent manner. To confirm this interpretation we separated the fractions from the HPLC sizing column by SDS-polyacrylamide gel electrophoresis and verified that only in the presence of ATP in both the premixture and column buffer did the DnaK and DnaJ proteins co-elute in these fractions (results not shown). The estimation of the apparent molecular weight of this putative DnaJ-DnaK complex suggests that it is slightly smaller than a dimer of DnaK. Thus, we conclude that a DnaJ dimer (or monomer) interacts with a DnaK monomer (Fig. 6).


Figure 6: Detection of a DnaK-DnaJ complex. The DnaK (35 µg) was incubated with DnaJ (40 µg) in the absence or presence of ATP in the premixture (DnaK, DnaJ, and ATP); in the presence of ATP both in the premixture and the column buffer (DnaK, DnaJ, and ATP/ATP) and in the presence of ATP in the premixture and ADP in the column buffer (DnaK, DnaJ, and ATP/ADP). Control experiments show the chromatography profile of either DnaK or DnaJ alone in the presence of ATP in the premixture and column buffer (DnaK, ATP/ATP and DnaJ, ATP/ATP, respectively). The concentration of ATP or ADP was 200 µM. The position of dimer DnaK (2K) was estimated by chromatographing the DnaK protein (10 mg/ml). At that concentration, the DnaK dimer form is easily detectable (Montgomery et al., 1993). The Superdex 200 column equilibrated with buffer B was calibrated with the following Bio-Rad molecular weight standards: 1, thyroglobulin (670 kDa); 2, bovine -globulin (158 kDa); 3, chicken ovalbumin (44 kDa); 4, equine myoglobin (17.5 kDa).



The substitution of ATP by ADP (or AMP-PCP) in the column buffer did not lead to the formation of a DnaK-DnaJ complex (result not shown), suggesting that the DnaK-ADP form derived from ATP hydrolysis is required for a stable DnaK-DnaJ interaction.


DISCUSSION

It was previously suggested that Hsp70 and DnaJ-like proteins (Hsp40) exhibit different substrate specificity (for review, see Cyr et al.(1994)). It is generally assumed that the Hsp70 class of protein binds to unfolded proteins, recognizing short polypeptides that are in an extended configuration. By contrast, it is believed that DnaJ-like proteins bind protein substrates in the ``molten globule'' state exhibiting secondary and some tertiary structures. While the binding of Hsp70 to different stretched polypeptides was carefully examined (Flynn et al., 1991; Blond-Elguindi et al., 1993a; Gragerov et al., 1994), no corresponding systematic study of binding of Hsp70 to a native protein substrate nor that of the DnaJ chaperone to native proteins or peptide substrates was done. In this paper we used four, native substrates, O, P, , and RepA, as well as a permanently unfolded polypeptide which lacks secondary structure and cannot fold, CMLA, to compare the ability of these substrates to form a complex with the DnaK or DnaJ molecular chaperones. We found that the DnaK protein, previously assumed to preferentially recognize unfolded peptides in the extended configuration, binds much better to native RepA then denatured CMLA (or heat inactivated luciferase).^4 Perhaps these seemingly conflicting observations with DnaK can be explained if one assumes that the RepA native protein exhibits (transiently?) a polypeptide loop(s) on the surface in the stretched configuration. On the other hand, DnaJ chaperone, previously supposed to recognize protein substrates with secondary and tertiary structure, indeed recognizes efficiently the native and RepA proteins but binds with very low affinity to either the native O, P proteins, or to the denatured CMLA. Perhaps and RepA exhibit such a protein structure on their surface that DnaJ likes to bind to. What amino acid sequence or tertiary structure is recognized by DnaJ protein remains still unknown.

In previously published studies it was assumed that ADP, ATP, and ATPS all bind to Hsp70 with comparable affinities, in the micromolar range (Palleros et al., 1991, 1993) This belief was recently challenged by other laboratories (Prasad et al., 1994; Montgomery et al., 1993). Taking into account the recently published data for the affinity of DnaK to ATP (K35 nM) and to ADP (K 200 nM) (Montgomery et al., 1993) most of DnaK (under physiological concentrations of these two nucleotides) would be in the nucleotide bound form. In the absence of the DnaJ protein, the DnaK in the presence of ATP possesses the highest affinity toward a substrate (high on rate), but exhibits an even higher off rate, as shown by using the stopped-flow technique (Schmid et al., 1994). In this paper we show that the presence of nonhydrolyzable ATP analogues can ``trap'' DnaK in a conformation that promotes efficient DnaK-substrate complex formation. However, we cannot exclude the possibility that for some protein substrates, binding to DnaK will also accelerate the ATP hydrolysis step and/or nucleotide exchange. Such a possibility was previously suggested for the eukaryotic Hsc70 by Hightower et al.(1994). It is also possible that in some cases, the binding of substrate to DnaK, which may induce conformational changes in the chaperone (Park et al., 1993), will also trigger the release of ATP or ADP from the DnaK chaperone.

Contrary to the previously published data (Palleros et al., 1991, 1994) we show in this paper that the presence of ADP in the premixture does not substantially accelerate the binding of DnaK to its protein substrate. Moreover, the hydrolyzable ATP leads to the dissociation of DnaK from substrate complex, as it was shown previously (Liberek et al., 1991b). We conclude that the DnaK-ADP form which is created after ATP hydrolysis possesses limited affinity to the protein substrates. As it will be discussed in the accompanying paper (Wawrzynów et al., 1995b), in order to stabilize DnaK-substrate complex, DnaK protein needs to be in the active conformation which can occur in the presence of DnaJ and ATP hydrolysis.

We previously showed that the presence of DnaJ accelerates (even without GrpE) the hydrolysis of ATP bound to DnaK (Liberek et al., 1991a; Szabo et al., 1994). In this paper we show that in the presence of hydrolyzable ATP it is possible to isolate DnaK-DnaJ complex. The presence of nonhydrolyzable ATP analogue, which promotes efficient binding of DnaK to its various protein substrates, does not stimulate DnaK-DnaJ complex formation. Our results suggest that DnaJ protein binds to the DnaK-ADP form only when this form is a product of DnaK-dependent ATP hydrolysis.

In summary, in this paper we show that the DnaK-ADP form possesses low affinity to the substrates but high affinity to the DnaJ protein. These results suggest that the mechanisms of DnaK binding to DnaJ and DnaK binding to substrate are fundamentally different. It is also possible that the DnaK protein domains which are involved in the interaction with a substrate and DnaJ are different. In preliminary experiments we have shown that a DnaK mutant variant, lacking 94 amino acids from its C-terminal domain (DnaKc94) does not interact stably with the DnaJ protein, but does form a functional (but unstable) complex with the P protein (this paper). (^6)These results suggest that DnaJ may interact stably with DnaK through DnaK's C-terminal domain. Such a possibility was previously suggested by Tsai and Wang(1994). However, more experiments are needed to verify this hypothesis. For example, it is still possible that the truncation of the C-terminal domain of the DnaK protein induces a conformational change of the N-terminal domain of DnaK protein, thus preventing formation of the DnaJ-DnaKc94 complex.


FOOTNOTES

*
This work was supported by Grant 6P203 042 06 from the Polish State Committee for Scientific Research. 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.

(^1)
The abbreviations used are: CMLA, carboxymethylated alpha-lactalbumin; ATPS, adenosine 5`-O-(3-thiotriphosphate); AMP-PNP, 5`-adenylyl beta,-imidodiphosphate; AMP-PCP, 5`-adenylyl beta,-methylenediphosphate; BSA, bovine serum albumin; PBS, phosphate-buffered saline: HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay.

(^2)
Ziemienowicz, A., Zylicz, M., Floth, C., and Hubsher, U.(1995) J. Biol. Chem., in press

(^3)
A. Wawrzynów and M. Zylicz, unpublished results.

(^4)
D. Wall, unpublished results.

(^5)
K. Liberek, unpublished results.

(^6)
A. Maddock, A. Wawrzynow, B. Banecki, D. Wall, C. Georgopoulos, and M. Zylicz, unpublished results.


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