©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of the ATPase Activity of the Molecular Chaperone DnaK by Peptides and the DnaJ and GrpE Heat Shock Proteins (*)

(Received for publication, October 12, 1994; and in revised form, December 15, 1994)

Robert Jordan (§) Roger McMacken (¶)

From the Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their K as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of DNA replication in vitro. The inhibitory effect of peptides on DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in DNA replication. Apparently, peptides sequester free DnaK protein and block DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the V(max) of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.


INTRODUCTION

One of the earliest observations of the involvement of cellular heat shock proteins and molecular chaperones in normal protein metabolism in unstressed cells came from genetic studies of bacteriophage DNA replication in Escherichia coli (for a review, see Friedman et al.(1) ). These investigations indicated that DnaK protein, the most abundant hsp70 homologue of E. coli, as well as the bacterial DnaJ and GrpE proteins were absolutely required for DNA replication at all temperatures. Characterization of the properties of conditional lethal dnaK, dnaJ, or grpE mutants of E. coli indicated that each mutant manifested similar phenotypes, such as (i) defects in the regulation of heat shock protein synthesis; (ii) reduced levels of general proteolysis; and (iii) defective RNA and DNA synthesis(2, 3) . Furthermore, several recent studies are consistent with the idea that the products of these three genes may also participate in protein folding in vivo(4, 5, 6) . These findings suggest that DnaK, DnaJ, and GrpE chaperonins functionally cooperate in several aspects of bacterial physiology.

The importance of both DnaK and DnaJ to cellular function is underscored by the presence of families of highly conserved homologues of these proteins in eukaryotic cells. Genetic, biochemical, and physiological studies of the functions of these Hsp70 family members and DnaJ-like proteins suggest that, as for prokaryotes, these proteins function in a diverse array of protein metabolic events (see Gething and Sambrook (7) and Craig et al.(8) for reviews). These processes include translocation of proteins across intracellular membrane barriers, protein folding and assembly, disaggregation of protein aggregates, disassembly of clathrin-coated vesicles during receptor-mediated endocytosis, as well as a complex cellular response to heat and certain other stress conditions. Some, if not all, of these events are apparently accomplished, at least in part, through a functional cooperation between specific hsp70 and DnaJ homologues(9, 10) .

Biochemical studies of the initiation of bacteriophage DNA replication in systems reconstituted with purified proteins have identified the stages where DnaK, DnaJ, and GrpE act in this process. DnaJ binds specifically to a prepriming nucleoprotein complex, assembled at the viral replication origin, that contains the O and P replication proteins and the E. coli DnaB helicase(11) . The bound DnaJ protein may destabilize this structure (12) and possibly plays a role in targeting DnaK to act on this preinitiation complex (11, 13) . In the presence of ATP and a large molar excess of DnaK, the preinitiation nucleoprotein structure is partially disassembled, resulting in release of entrapped DnaB helicase, which, in turn, eventuates in the initiation of DNA replication(13, 14, 15, 16) . The GrpE cochaperonin is absolutely required for DNA replication in vivo but is not essential for replication in vitro at elevated DnaK concentrations. Regardless, GrpE does increase the efficiency of DnaK action in the reconstituted replication system. In the presence of GrpE, the concentration of DnaK required for DNA replication is reduced 5-10-fold(13, 15) .

We were interested in defining the mechanism by which the DnaK molecular chaperone and the DnaJ and GrpE cochaperonins mediate the ATP-dependent disassembly of nucleoprotein preinitiation complexes during the initiation of DNA replication. Our initial mechanistic studies of this phenomenon, however, were hampered by the extreme complexity of the preinitiation nucleoprotein structure, which may contain upwards of 40 separate polypeptide chains. We decided to test the notion that simple protein ``substrates'' could be utilized both to probe DnaK function and to study the mechanisms by which the DnaJ and GrpE cochaperonins activate the weak intrinsic ATPase activity of DnaK(17) . Since DnaK and other Hsp70 family members such as hsc70 and BiP are known to interact ``nonspecifically'' with various short peptides(18, 19, 20, 21) , we explored the possibility that peptides might serve as suitable alternate effectors of the intrinsic ATPase activity of DnaK.

In this report, we demonstrate that peptides containing at least 6 amino acid residues stimulate the intrinsic ATPase of DnaK. We show that those peptides that most effectively activate the ATPase of DnaK behave as potent inhibitors of DNA replication in vitro. We conclude that the peptide-dependent ATPase activity of DnaK provides an attractive model system for studying the mechanism of action of this molecular chaperone. Accordingly, we make use of this system to examine the influence of the DnaJ and GrpE cochaperonins on the peptide-dependent and peptide-independent ATPase activities of DnaK.


EXPERIMENTAL PROCEDURES

Reagents and Materials

Materials and their sources were Hepes (Research Organics Co.); ATP-agarose (C-8 linkage), potassium glutamate, lithium chloride, and magnesium acetate (Sigma); formic acid, PEI-cellulose (^1)TLC sheets (20 times 20 cm) (EM Science); [2,8-^3H]ATP (30-50 Ci/mmol) and [alpha-P]ATP (geq3000 Ci/mmol) (Amersham Corp.); ATP and MonoQ chromatography columns (Pharmacia Biotech Inc.); and Centricon spin concentrators (Amicon Corp.).

Bacteriophage and E. coli Replication Proteins

The purification methods and specific activities of many of the proteins required for bacteriophage DNA replication have been described elsewhere(22) . DnaK protein was purified to homogeneity by a modification of the previously published method(22) . The acid precipitation step, involving dialysis of the resuspended ammonium sulfate pellet against a sodium acetate buffer, was omitted. DnaK-containing fractions from the ATP-agarose column, containing 27 mg of protein, were dialyzed against 4 liters of buffer T (25 mM Hepes/NaOH, pH 7.6, 10% (v/v) glycerol, 1 mM EDTA, and 1 mM dithiothreitol) and were further purified by fast protein liquid chromatography on a MonoQ column. The entire sample was applied to a MonoQ 10/10 column equilibrated in buffer T. The column was washed with 40 ml of buffer T, and DnaK was eluted with a 180-ml linear gradient of 0-0.6 M NaCl in buffer T at a flow rate of 1 ml/min. Fast protein liquid chromatography on MonoQ removed free ATP as well as all nucleotide (both ADP and ATP) bound to DnaK. This assessment is based on the ratio of absorbances of the DnaK preparation at 260 nm to that at 280 nm and by a demonstration that prebound radiolabeled nucleotide ([alpha-P]ATP) is completely removed from DnaK during chromatography on MonoQ (data not shown). The peak fractions (22 ml, 15 mg of DnaK) were dialyzed against 4 liters of buffer H (25 mM Hepes/KOH, pH 7.6, 0.1 M potassium glutamate, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol). The DnaK preparation was quick-frozen in liquid nitrogen and stored at -85 °C.

GrpE protein was purified to homogeneity from E. coli strain C600dnaK103 by a modification of a published procedure(23) . Following chromatography of GrpE on a DnaK affinity column and its elution by ATP, fractions containing GrpE (25 ml) were concentrated, and the buffer was exchanged to buffer H with 2 mM dithiothreitol using Centricon spin concentrators. This procedure removed ATP from the GrpE preparation, which was quick-frozen and stored at -85 °C.

Synthesis and Purification of Peptides

Peptides A, C, and I have been previously described(18) . Peptide A, peptide X, and soluble mixtures of random peptides of various defined lengths were generously donated by G. Flynn and J. Rothman (Memorial Sloan-Kettering Cancer Center, NY). Preparation of these peptides has been described(18, 19, 24) . Peptide C and peptide I were synthesized by solid phase peptide synthesis by the Johns Hopkins School of Medicine Protein/Peptide Facility. Peptides C and I were further purified by reverse-phase chromatography in 0.03% trifluoroacetic acid on C18 high pressure liquid chromatography columns. Peptides were eluted with a 0-60% acetonitrile gradient. Peptide concentrations were determined by ninhydrin assays or by direct weighing of freshly lyophilized powder. The amino acid sequences of the aforementioned peptides are peptide C, KLIGVLSSLFRPK; peptide I, SNGSLQCRIC; peptide A, KRQIYTDLEMNRLGK; and peptide X, KFERQ.

ATPase Assay

Unless otherwise stated, the ATPase reaction mixture (25 µl) contained the following components: 25 mM Hepes/KOH, pH 7.6, 0.1 M potassium glutamate, 11 mM magnesium acetate, 0.67 µM DnaK, 80 µM ATP, and 4 µCi [^3H]ATP. The reaction mixture was incubated at 30 °C, and 1-µl portions were applied to PEI-cellulose thin layer chromatography plates at appropriate times. The PEI-cellulose plates were pre-spotted with 1 µl of a mixture containing 20 mM ATP and 20 mM ADP. The reaction rates were equivalent when the reactions were terminated by lowering the pH of the reaction mixture to pH 3 with HCl or terminated by spotting the reaction mixture directly onto a thin layer plate. The PEI-cellulose plates were developed in 1 M formic acid, 0.5 M LiCl. The location of the radiolabeled ATP and ADP was determined by marking the location of unlabeled ATP and ADP standards visualized by fluorescence quenching using short wave ultraviolet irradiation. The sections of the plate containing nucleotide were placed (matrix side up) in a 20-ml scintillation vial, covered with 6 ml of CytoScint scintillation fluid (ICN Biochemicals), and counted in a liquid scintillation spectrometer. For each reaction condition, at least five samples were taken in the linear range of the assay. Accordingly, the amount of ATP hydrolysis was not permitted to exceed 20% of the amount of ATP initially present. The kinetic data were analyzed with Enzfitter (Biosoft, Cambridge, United Kingdom), a non-linear regression program.

DNA Replication Assays

Replication of plasmid templates that contain the bacteriophage origin of replication was performed as previously described(22) . Replication of single-stranded M13 mp9 DNA was performed essentially as described(25) . The reaction mixture was assembled in two stages. The first stage mixture (15 µl) contained 40 mM Hepes/KOH, pH 7.6, 11 mM magnesium acetate, 300 mM potassium glutamate, 100 ng of rifampicin, 4 mM ATP, 160 ng of M13 mp9 single-stranded DNA, 260 ng of DnaB protein, and 200 ng of primase. The second-stage reaction mixture contained 166 µM each of CTP, GTP, and UTP; 360 µM each of dATP, dCTP, and dGTP; 160 µM dTTP, with [methyl-^3H]dTTP at 100-150 cpm/pmol of total deoxynucleotide; 350 ng of E. coli single-stranded DNA-binding protein, and 25 ng of DNA polymerase III holoenzyme. The first-stage reaction mixture was assembled on ice. It was incubated at 30 °C for 5 min and then supplemented with 15 µl of the second-stage reaction mixture. The combined mixture was incubated for an additional 10 min at 30 °C. DNA synthesis was measured by determining the level of incorporation of labeled deoxynucleotide into acid-insoluble material, which was collected on a glass fiber filter (Whatman 934-AH) and counted in a liquid scintillation counter.

Determination of Protein Concentrations

The concentrations of DnaJ and GrpE were determined by a modification of the method of Lowry et al.(26) using bovine serum albumin as a standard. The concentration of DnaK was determined by quantitative amino acid composition analysis performed at the Johns Hopkins School of Medicine Protein Peptide Facility using the PICO-TAG Amino Acid Analysis system developed by Millipore-Waters. The concentration of subsequent preparations of nucleotide-free DnaK were determined using the calculated molar extinction coefficient of the native protein. The molar extinction coefficient of native DnaK, determined by the method of Gill and von Hippel(27) , is 15,800 M cm.


RESULTS

The ATPase Activity of DnaK Is Stimulated by Small Peptides

E. coli DnaK protein functions in the initiation of bacteriophage DNA replication by mediating the ATP-dependent disassembly of a nucleoprotein preinitiation complex that is assembled at the viral replication origin(15, 16, 28) . Analysis of the mechanism of DnaK action in this process is impeded by the complexity and large size of the preinitiation complex, which is composed of six different proteins and at least 25 polypeptide chains. We initially sought, therefore, to learn more about the interaction of DnaK with less complex substrates. Flynn et al.(18) had demonstrated that certain small peptides, including peptides A, C, and I, stimulate the intrinsic ATPase activity of two members of the eukaryotic Hsp70 family, hsc70 protein and BiP. Given that DnaK shares significant amino acid sequence homology with these proteins, it seemed likely that small peptides might serve as suitable substrates/effectors for studies of DnaK function.

Each of several different peptides was found to stimulate the weak intrinsic ATPase activity of DnaK (Fig. 1) as measured under steady-state conditions. In the absence of peptide, the turnover number for ATP hydrolysis by DnaK is approximately 0.02-0.04/min, depending on the preparation of DnaK used. ATP hydrolysis rates by DnaK were stimulated 5-20-fold by the presence of saturating levels of peptide. The amount of stimulation depended on the amino acid sequence of the peptide effector, with peptide I providing significantly more stimulation of DnaK ATPase activity than either peptides A or C. Additionally, the concentrations of peptide that produced half maximal activation of the DnaK ATPase activity (K(A)) were different for each peptide (Table 1). Peptide X, which is known to interact with at least one member of the eukaryotic Hsp70 protein family(24) , did not stimulate the intrinsic ATPase activity of DnaK (data not shown), even at millimolar concentrations, and was used as a negative control peptide in subsequent experiments.


Figure 1: Small peptides stimulate the ATPase activity of DnaK. The peptide-dependent ATPase assay was performed as described under ``Experimental Procedures.'' Each reaction mixture contained 16.7 pmol of DnaK protein. Peptide was added at the indicated concentrations to the individual reaction mixtures. Opencircles, peptide C; closedcircles, peptide A; closedsquares, peptide I. All data points have been corrected for the amount of peptide-independent ATP hydrolysis by DnaK (0.55 pmol/min).





Inhibition of Bacteriophage DNA Replication by Peptides

The observation that binding of peptides to DnaK stimulates its inherent ATPase activity indicates that the peptide-binding site(s) on DnaK may play an integral role in the action of this molecular chaperone in the initiation of phage DNA replication. If so, peptides that bind tightly to DnaK might serve as effective inhibitors of DnaK function in DNA replication. Persistent occupation of the peptide-binding site of DnaK with nonspecific peptides, such as peptide C, would be expected to divert DnaK away from productive interactions with specific protein substrates. Peptides at high concentrations might therefore block the DnaK molecular chaperone from mediating the ATP-dependent protein disassembly reactions required for initiation of DNA replication. Consistent with this idea, a total inhibition of DNA replication in vitro was observed when a reconstituted multiprotein replication system was supplemented with 1 mM peptide C (Fig. 2). A 50% reduction in replication activity was obtained at 125 µM peptide C. Other peptides that stimulate the innate ATPase activity of DnaK, such as peptides A and I, also effectively inhibited initiation of DNA replication in vitro; 50% reduction of DNA replication occurred at 350 µM peptide A and 190 µM peptide I (data not shown).


Figure 2: Inhibition of bacteriophage DNA replication in vitro by nonspecific peptides. DNA replication was carried out as described under ``Experimental Procedures,'' except that each reaction mixture contained 4.3 µg of DnaK and either peptide C (closedcircles) or peptide X (closedtriangles). In a separate set of reactions, the replication reaction mixtures contained DnaK (0.85 µg), GrpE (35 ng), and peptide C (opencircles). 100% DNA synthesis represents the amount of DNA replication obtained in the absence of added peptide (600 pmol).



The results of several control experiments indicated that, of the nine proteins that constitute the in vitro replication system, DnaK protein apparently is the specific target of the peptide inhibitors. For example, only those peptides that activate DnaK ATPase activity act as inhibitors of DNA replication in vitro. Peptide X neither stimulates the intrinsic ATPase activity of DnaK nor affects the activity of the reconstituted replication system significantly (Fig. 2). Furthermore, addition to the replication system of GrpE, a cochaperonin of DnaK, greatly reduced the inhibitory effect of high concentrations of peptide C (Fig. 2), even though 5-fold less DnaK was present in the replication mixture. GrpE is known to increase the efficiency of DnaK action in DNA replication (13, 15) and to stimulate the peptide-dependent ATPase activity of DnaK (see below). More direct evidence that DnaK is the target of peptide-mediated inhibition of DNA replication is the finding that the inhibition rendered by 250 µM peptide C is largely neutralized when the concentration of DnaK present in the replication system is increased (Fig. 3). Finally, high concentrations of peptide C had no effect on the in vitro replication of a single-stranded DNA template, M13 mp9, a reaction that does not require the presence of DnaK, DnaJ, or GrpE (data not shown). This latter system did, however, require several of the E. coli replication proteins that participate in the propagation of replication forks during DNA replication, such as DnaB helicase, DnaG primase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. Taken together, the foregoing results are all consistent with the conclusion that binding of peptide to DnaK is responsible for the peptide-mediated inhibition of DNA replication observed in Fig. 2. These studies suggest that nonspecific peptides interact with DnaK in a fashion qualitatively similar to certain physiological protein ``substrates'' of DnaK. We conclude that peptides most likely will serve as useful analogues of protein substrates and will permit detailed characterization of the effects of DnaJ and GrpE on the peptide-dependent and independent ATPase activities of DnaK.


Figure 3: Peptide-mediated inhibition of bacteriophage DNA replication is diminished at high concentrations of DnaK. DNA replication was carried out in vitro as described under ``Experimental Procedures,'' except that the amount of DnaK present in the reaction mixture was varied as indicated. The reaction mixtures contained either no added peptide (closedcircles) or 250 µM peptide C (opencircles).



Effect of Peptide Length on the ATPase Activity of DnaK

We investigated the chain length dependence of the peptide-binding site of DnaK. Peptides of random sequence but defined length were tested at a concentration of 500 µM for their capacity to stimulate the intrinsic ATPase activity of DnaK. Short peptides, containing 5 amino acid residues or less, failed to stimulate DnaK ATPase activity under the conditions tested (Fig. 4). Random sequence peptides with lengths geq 6 amino acid residues, however, did serve as effectors of the ATPase activity of DnaK. Maximal stimulation of DnaK ATPase activity required peptides at least 8 amino acids in length (Fig. 4). A similar response of the DnaK ATPase to peptide length was obtained when DnaK was supplemented with the DnaJ and GrpE cochaperonins (Fig. 4).


Figure 4: Effect of peptide length on the peptide-dependent ATPase activity of DnaK. The ATPase assay was performed as described under ``Experimental Procedures,'' except that each reaction mixture was supplemented with random peptides. The random peptides, containing the indicated number of amino acid residues, were each present at a concentration of 500 µM. All data have been corrected for the intrinsic peptide-independent ATPase activity of DnaK (0.55 pmol of ATP hydrolyzed/min). Opencircles, DnaK (0.67 µM) alone; closedsquares, DnaK (0.67 µM) supplemented with DnaJ (0.12 µM) and GrpE (0.3 µM).



Effects of GrpE on Peptide-dependent DnaK ATPase Reaction

The GrpE cochaperonin has no intrinsic ATPase activity, but it does stimulate the rate of hydrolysis of ATP by DnaK when high levels of peptide effectors are present in the reaction mixture (Fig. 5). In the absence of peptides, GrpE has no effect on the weak intrinsic ATPase activity of DnaK ( (17) and data not shown). The concentration of GrpE that provides nearly optimal stimulation of the DnaK ATPase is approximately 0.1 µM, regardless of which peptide effector is present (Fig. 5). Additional studies indicated that the concentration of GrpE required to maximally activate the ATPase activity of DnaK is independent of the concentration of DnaK. A GrpE concentration of geq0.1 µM was needed for maximal ATPase activity even when the DnaK concentration was reduced severalfold (data not shown). It also might be notable that when GrpE is present, the V(max) for ATP hydrolysis by DnaK depends on the peptide sequence.


Figure 5: Effect of GrpE protein on the peptide-dependent ATPase of DnaK. The ATPase assay was performed as described under ``Experimental Procedures,'' except that each reaction mixture contained GrpE protein at the indicated concentration. Additionally, the reaction mixtures contained 1.2 mM peptide A (closedcircles), 1 mM peptide C (opencircles), or 1.5 mM peptide I (closedsquares).



Effect of DnaJ on the ATPase Activity of DnaK

DnaJ has no intrinsic ATPase activity and does not affect the peptidedependent ATPase activity of DnaK when saturating levels of peptide C are present, even at an 8-fold molar ratio of DnaJ to DnaK (Fig. 6). Nevertheless, DnaJ is a potent effector of the DnaK ATPase when peptide is absent. Under these conditions, the rate of ATP hydrolysis by DnaK increases approximately in a linear fashion as the concentration of DnaJ is raised (Fig. 6), reaching a maximal rate at a DnaJ concentration of 0.76 µM. A 6-fold molar ratio of DnaJ to DnaK produced an ATPase rate equivalent to that given by saturating concentrations of peptide C (Fig. 6). It may be significant that higher concentrations of DnaJ but not of peptide C were inhibitory on DnaK ATPase activity (data not shown).


Figure 6: Effects of DnaJ on the ATPase activity of DnaK. The ATPase assay was performed as described under ``Experimental Procedures'' with the following modifications. Each reaction mixture contained 0.13 µM DnaK, DnaJ protein, as indicated, and either 1 mM peptide C (closedcircles) or no peptide C (opencircles).



Effects of DnaJ and GrpE on the Peptide-dependent ATPase of DnaK

We kinetically examined the effects of DnaJ and GrpE on the peptide-dependent activation of the ATPase activity of DnaK at a saturating concentration of ATP. The amounts of ATP hydrolyzed in the presence of various combinations of DnaK and its cochaperonins were measured at varying (subsaturating) concentrations of peptide C. Similar experiments have been performed with peptides A and I. Fig. 7depicts a standard replot of the kinetic data for peptide C. The intercepts on the ordinate provide an estimate of the V(max) for ATP hydrolysis, and the slopes of the individual plots provide an estimate of the K(A) for peptide C. The K(A) is defined, in these experiments, as the concentration of peptide C that yields half-maximal activation of the ATPase activity of DnaK.


Figure 7: Effects of DnaJ and/or GrpE on the kinetics of ATP hydrolysis by DnaK in the presence of varying concentrations of peptide effector. The ATPase assays were performed with 0.67 µM DnaK as described under ``Experimental Procedures'' but with the following modifications. Each reaction mixture contained a subsaturating concentration of peptide C, which was varied over the range of 20-800 µM. Additionally, each mixture was supplemented with 0.13 µM DnaJ (opencircles), 0.22 µM GrpE (closedsquares), 0.13 µM DnaJ and 0.22 µM GrpE (opentriangles), or had no further supplements (closedcircles).



Table 1provides a summary of the kinetic constants obtained with each of the three peptides. In the presence of GrpE, the maximal rate of peptide-dependent ATP hydrolysis by DnaK increased significantly (see also Fig. 5). Moreover, the apparent K(A) for peptide increased approximately 2-3-fold when GrpE was present. In contrast, when DnaK was supplemented with DnaJ, the K(A) for peptide decreased 2-4-fold, but the maximal rate of ATP hydrolysis remained unchanged or decreased slightly. When both cochaperonins were present with DnaK, the V(max) of ATP hydrolysis increased, and the K(A) for peptide decreased relative to the reactions containing DnaK alone. Thus, when all three proteins were present, the effect of DnaJ to lower peptide K(A) was dominant over the effect of GrpE to increase K(A), whereas the effect of GrpE to increase V(max) was observed even when DnaJ was also present.


DISCUSSION

The stimulation of the intrinsic ATPase activity of DnaK by peptides reported here shares several features with the peptide-dependent activation of the ATPase activities of eukaryotic hsp70 homologues such as bovine hsc70 and BiP(18, 19) . Each of these proteins has a very weak intrinsic ATPase activity with turnover numbers ranging from 0.02 to 0.04 min, depending on the preparation. The ATPases of all three proteins are activated 5-20-fold by saturating concentrations of peptide effector. The prokaryotic and eukaryotic hsp70 molecular chaperones also respond in a similar fashion to peptide length ( Fig. 4and (19) ). For both DnaK and BiP, peptides of random sequence shorter than 5 amino acid residues in length fail to stimulate the chaperone's intrinsic ATPase. Furthermore, maximal activation of the ATPase associated with each of these hsp70 chaperones occurs when the peptide effector contains at least 8 residues. A qualitatively similar peptide-length dependence was found for the DnaK ATPase when the DnaJ and GrpE cochaperonins, both essential for DnaK function in vivo, were also present in the reaction mixture.

We have shown that the DnaJ cochaperonin, like short peptides, stimulates the ATPase activity of DnaK. However, the K(A) for DnaJ, 0.2-0.3 µM, is approximately 1000-fold lower than the K(A) for an average random peptide. The lower K(A) suggests that DnaJ interacts more effectively with DnaK than do peptides, but the DnaK-DnaJ interaction is still too weak to be detected by standard chromatographic methods. (^2)Previously, it had been determined that DnaJ alone did not have the capacity to activate the DnaK ATPase(17) , but a more recent study, published after the completion of this work, indicates that DnaJ does indeed activate the DnaK ATPase(29) . There is evidence that the capacity of DnaJ to activate the intrinsic ATPase of an hsp70 protein has been conserved during evolution. The YDJ1 protein, a cytoplasmic homologue of DnaJ protein found in budding yeast, has been shown to activate the intrinsic ATPase activity of SSA1 protein, a DnaK homologue and Hsp70 family member localized in the cytoplasm of this organism(30) .

In the presence of a saturating concentration of peptide, DnaJ has no influence on the ATPase activity of DnaK (Fig. 6). One interpretation of this finding is that DnaJ and peptide compete for binding to the same site on DnaK. Perhaps DnaJ contains a segment of extended polypeptide that interacts with the peptide-binding site of DnaK. Preliminary experiments, in fact, indicate that DnaJ does contain a glycine-rich and phenylalanine-rich segment (amino acids 76-105) that is highly sensitive to proteolytic digestion, which implies that this region of the cochaperonin exists in an extended conformation in solution. (^3)Furthermore, studies of DnaJ deletion mutant proteins suggest that this segment of DnaJ appears to play an important role in the capacity of DnaJ to activate the DnaK ATPase. There is, however, a second possible explanation for the failure of DnaJ to activate the DnaK ATPase when saturating concentrations of peptide are present. This interpretation is based on findings gathered from a comprehensive analysis of the kinetics of the DnaK ATPase reaction that we have undertaken. (^4)We have determined that DnaJ and peptide each stimulate the rate-limiting step in the DnaK ATPase reaction. However, when peptide is present at high concentration, we have discovered that a different step in the DnaK ATPase cycle now becomes rate limiting. Since DnaJ is not capable of stimulating the rate of this new rate-limiting step, it has no effect on the steady-state rate of ATP hydrolysis by DnaK under these conditions.

Small peptides at high concentrations are strong inhibitors of a multienzyme system that supports the initiation and propagation of phage DNA replication. The peptide-mediated inhibition is not permanent. The inhibitory effects of peptides on DNA replication can be countered either by raising the concentration of DnaK in the multiprotein in vitro system or by including small amounts of GrpE, which has been shown to increase the effectiveness of DnaK action(13, 15) . These findings suggest that DnaK is either the direct target of peptide inhibition or is required for relieving the inhibition of peptide on the function of some other protein in the replication system. Two additional results are consistent with the view that DnaK itself is the target of peptide-mediated inhibition. First, there is a strong correlation between the K(A) of a peptide for stimulation of the ATPase activity of DnaK and its apparent K(I) for inhibition of DNA replication in the reconstituted multiprotein system. Thus, those peptides that have the highest apparent affinity for DnaK act as the most potent inhibitors of DNA replication. Second, peptides had no effect on the replication of a single-stranded DNA template in an in vitro system composed of DnaB helicase, primase, E. coli single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. Since these same four E. coli replication proteins also participate in the propagation of replication forks on the chromosome, we presume that peptides block the function of a protein that acts during the initiation phase of DNA replication. Of the proteins involved in initiation of DNA replication, which include DnaJ, DnaK, and the O and P proteins, only DnaK is known to interact with short peptides.

Taken together these results suggest that peptides competitively inhibit DNA replication by binding to free DnaK and occupying the single polypeptide chain binding site on this molecular chaperone. This would divert DnaK from productive interactions with the multiprotein preinitiation complex that is formed at the origin. It is possible that the presence of peptides in the in vitro replication system establishes an environment that more closely mimics the physiological conditions found during DNA replication in vivo, where high intracellular concentrations of protein may in fact sequester most, if not all, of the available DnaK. This hypothesis could explain why GrpE protein is required for DNA replication in vivo. As shown in this report, GrpE increases the efficiency of ATP hydrolysis by DnaK when peptides or protein effectors are present, possibly by directly or indirectly accelerating the release of bound polypeptides from DnaK. Thus, in this scenario, GrpE acts to increase the level of free DnaK in the cell, making more molecules of DnaK available for productive interactions with specific protein substrates, such as the preinitiation nucleoprotein structures that form at the replication origin during the initiation of DNA replication.

It is also possible that GrpE improves the selectivity of DnaK interaction with polypeptide substrates. In support of this idea, Zylicz and colleagues(31) , on the basis of a cross-linking analysis, have concluded that DnaJ and GrpE increase the affinity of DnaK for the P replication protein. Our kinetic analysis of the peptide-stimulated ATPase activity of DnaK indicates that the presence of DnaJ alone yields a lower K(A) for all peptides tested (Table 1). This suggests that DnaJ may increase the affinity of DnaK for peptides. However, this is not a certainty, since the K(A) for peptide is a kinetic constant, and it is unlikely that it will prove to be equivalent to the equilibrium dissociation constant, K(D), for peptide binding to DnaK. In contrast to the action of DnaJ, GrpE serves to raise the K(A) of peptides in the DnaK ATPase reaction (Table 1). When both DnaJ and GrpE are present with DnaK, the DnaJ effect on peptide K(A) predominates over that of GrpE, such that the K(A) for peptide-mediated stimulation of the DnaK ATPase is lower than it is with DnaK alone.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM36526 from the U. S. Public Health Service. 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.

§
Present address: Dana Farber Cancer Institute, Rm. B430, 44 Binney St., Boston, MA 02115.

To whom all correspondence should be addressed: Dept. of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3949; Fax: 410-955-2926.

(^1)
The abbreviation used is: PEI, polyethyleneimine.

(^2)
R. Jordan and R. McMacken, unpublished data.

(^3)
A. W. Karzai and R. McMacken, manuscript in preparation.

(^4)
A. Mehl and R. McMacken, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank G. Flynn and J. Rothman for generous gifts of peptides A and X and of random sequence peptides of defined chain length. We also thank A. Mehl, A. W. Karzai, and R. Russell of this laboratory for helpful discussions and for critically reviewing this manuscript.


REFERENCES

  1. Friedman, D. I., Olson, E. R., Georgopoulos, C., Tilly, K., Herskowitz, I., and Banuett, F. (1984) Microbiol. Rev. 48, 299-325
  2. Georgopoulos, C., Ang, D., Liberek, K., and Zylicz, M. (1990) in Stress Proteins in Biology and Medicine (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 191-221, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  3. Gross, C. A., Straus, D. B., Erickson, J. W., and Yura, T. (1990) in Stress Proteins in Biology and Medicine (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 167-189, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  4. Schroder, H., Langer, T., Hartl, F. U., and Bukau, B. (1993) EMBO J. 12, 4137-4144 [Abstract]
  5. Ziemienowicz, A., Skowyra, D., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 25425-25431 [Abstract/Free Full Text]
  6. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F. U. (1992) Nature 356, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  7. Gething, M. -J., and Sambrook, J. (1992) Nature 355, 33-44 [CrossRef][Medline] [Order article via Infotrieve]
  8. Craig, E. A., Gambill, B. D., and Nelson, R. J. (1993) Microbiol. Rev. 57, 402-414 [Abstract]
  9. Scidmore, M. A., Okamura, H. H., and Rose, M. D. (1993) Mol. Biol. Cell 4, 1145-1159 [Abstract]
  10. Feldheim, D., Rothblatt, J., and Schekman, R. (1992) Mol. Cell. Biol. 12, 3288-3296 [Abstract]
  11. Alfano, C., and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708 [Abstract/Free Full Text]
  12. Hoffmann, H. J., Lyman, S. K., Lu, C., Petit, M. A., and Echols, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12108-12111 [Abstract]
  13. Zylicz, M., Ang, D., Liberek, K., and Georgopoulos, C. (1989) EMBO J. 8, 1601-1608 [Abstract]
  14. Dodson, M., Echols, H., Wickner, S., Alfano, C., Mensa-Wilmot, K., Gomes, B., LeBowitz, J., Roberts, J. D., and McMacken, R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7638-7642 [Abstract]
  15. Alfano, C., and McMacken, R. (1989) J. Biol. Chem. 264, 10709-10718 [Abstract/Free Full Text]
  16. Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725 [Abstract/Free Full Text]
  17. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2874-2878 [Abstract]
  18. Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390 [Medline] [Order article via Infotrieve]
  19. Flynn, G. C., Pohl, J., Flocco, M. T., and Rothman, J. E. (1991) Nature 353, 726-730 [CrossRef][Medline] [Order article via Infotrieve]
  20. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 355, 455-457 [CrossRef][Medline] [Order article via Infotrieve]
  21. Gragerov, A., Zeng, L., Zhao, X., Burkholder, W., and Gottesman, M. E. (1994) J. Mol. Biol. 235, 848-854 [CrossRef][Medline] [Order article via Infotrieve]
  22. Mensa-Wilmot, K., Seaby, R., Alfano, C., Wold, M. S., Gomes, B., and McMacken, R. (1989) J. Biol. Chem. 264, 2853-2861 [Abstract/Free Full Text]
  23. Zylicz, M., Ang, D., and Georgopoulos, C. (1987) J. Biol. Chem. 262, 17437-17442 [Abstract/Free Full Text]
  24. Chiang, H.-L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989) Science 246, 382-385 [Medline] [Order article via Infotrieve]
  25. LeBowitz, J. H., Zylicz, M., Georgopoulos, C., and McMacken, R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3988-3992 [Abstract]
  26. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  27. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326 [Medline] [Order article via Infotrieve]
  28. Liberek, K., Georgopoulos, C., and Zylicz, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6632-6636 [Abstract]
  29. Wall, D., Zylicz, M., and Georgopoulos, C. (1994) J. Biol. Chem. 269, 5446-5451 [Abstract/Free Full Text]
  30. Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931 [Abstract/Free Full Text]
  31. Osipiuk, J., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 4821-4827 [Abstract/Free Full Text]

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