(Received for publication, October 12, 1994; and in revised form, December 15, 1994)
From the
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
of ATP hydrolysis by DnaK was elevated approximately 40-fold by
the presence of GrpE and saturating levels of peptides.
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.
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.
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) 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).
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).
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).
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).
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).
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 for peptide increased approximately
2-3-fold when GrpE was present. In contrast, when DnaK was
supplemented with DnaJ, the K
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
of ATP hydrolysis
increased, and the K
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
was dominant over the effect of GrpE to increase K
, whereas the effect of GrpE to increase V
was observed even when DnaJ was also present.
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 for DnaJ, 0.2-0.3 µM, is
approximately 1000-fold lower than the K
for an
average random peptide. The lower K
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. (
)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. ()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. (
)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
of a peptide for
stimulation of the ATPase activity of DnaK and its apparent K
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
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
for peptide is a kinetic constant, and it is
unlikely that it will prove to be equivalent to the equilibrium
dissociation constant, K
, for peptide binding to
DnaK. In contrast to the action of DnaJ, GrpE serves to raise the K
of peptides in the DnaK ATPase reaction (Table 1). When both DnaJ and GrpE are present with DnaK, the
DnaJ effect on peptide K
predominates over that of
GrpE, such that the K
for peptide-mediated
stimulation of the DnaK ATPase is lower than it is with DnaK alone.