(Received for publication, July 5, 1995)
From the
DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication
initiator protein of plasmid P1 in a reaction dependent on ATP and
Mg. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 µM free Mg
. In this paper, we demonstrate that GrpE
lowers the requirement of DnaK ATPase for Mg
,
resulting in a large stimulation of ATP hydrolysis at about 1
µM Mg
with and without DnaJ and RepA. In
contrast to its effect on the Mg
requirement, GrpE
increases the ATP requirement for DnaK ATPase and dramatically lowers
the affinity of DnaK for ATP in the absence of Mg
. We
propose that GrpE not only lowers the affinity of DnaK for nucleotide
but, by increasing affinity of DnaK for Mg
, also
weakens the interactions of Mg
with nucleotide prior
to its release.
Molecular chaperones from the 70-kDa heat shock protein (Hsp70) ()family are universally conserved ATPases, present in all
prokaryotic and eukaryotic cells (1, 2, 3) .
Although the synthesis of Hsp70s is transiently stimulated in response
to stress, most organisms synthesize them or related proteins, 70-kDa
heat shock cognates (Hsc70s), constitutively in non-stress conditions.
For normal growth, these molecular chaperones are required for folding
newly synthesized polypeptides, translocating polypeptides through
membranes, proteolysis, disassembly of clathrin-coated vesicles, and
DNA replication of several phage and plasmids. During heat shock,
Hsp70s prevent aggregation of heat-inactivated proteins, dissociate
protein aggregates formed by heat, and regulate the heat-shock
response.
The most abundant Escherichia coli Hsp70 homolog,
DnaK, acts with two other highly conserved heat shock proteins, DnaJ
and GrpE. Mutations in dnaK, dnaJ, and grpE have similar pleiotropic effects on E. coli growth and
physiology of several phage and plasmids. Furthermore, DnaK, DnaJ, and
GrpE function together in several in vitro systems, including
DNA replication of plasmid P1 and phage (4, 5, 6) and reactivation of
heat-inactivated RNA polymerase(7) , luciferase(8) ,
and rhodanese(9) . In all of those systems, DnaJ tags selected
native proteins or unfolded polypeptides for recognition by DnaK and
stimulates the hydrolysis of DnaK-bound ATP. GrpE binds DnaK tightly
through a conserved loop in the ATPase domain of DnaK near the
nucleotide binding site (10) and releases nucleotides from
DnaK(11) . The stimulation of the nucleotide release from DnaK
by GrpE results in increased DnaK ATPase activity and more efficient
recycling of DnaK. Homologs of DnaJ and GrpE have been found in many
eukaryotic and prokaryotic cells, suggesting that those two heat shock
proteins are required for many, if not all, biological functions of
Hsp70s and Hsc70s(12, 13, 14, 29) .
In plasmid P1 replication DnaK, DnaJ, and GrpE activate site-specific DNA binding by the replication initiator protein, RepA(15, 16) . RepA activation in vitro with purified proteins results from the conversion of RepA dimers to monomers(17, 18, 19) . RepA dimers are inactive for specific DNA binding, but they form a complex with DnaJ dimers that then can be recognized by DnaK. DnaK in an ATP-dependent reaction dissociates the RepA-DnaJ complex such that RepA is released in a monomeric form. RepA monomers bind avidly to specific DNA sequences within the P1 plasmid origin.
In vitro, the RepA
activation reaction can be efficiently catalyzed by DnaJ and DnaK
without GrpE(17, 18) . We discovered that the
requirement for GrpE emerges specifically when the free Mg concentration is lowered to about 1 µM(20) .
In this paper, we demonstrate that GrpE increases the affinity of DnaK
ATPase for Mg
, resulting in the stimulation of both
DnaK ATPase and RepA activation at about 1 µM
Mg
. We show also that GrpE decreases the affinity of
DnaK for ATP and discuss the possibility that the nucleotide release
from DnaK by GrpE requires not only the lowering of DnaK affinity for
nucleotide but also the weakening of the interactions of nucleotide
with Mg
.
[H]oriP1 DNA (an 1186-base pair DNA
fragment containing five RepA binding sites in the P1 origin) was
prepared as described(17) .
Figure 1:
GrpE effects on RepA activation by DnaK
and DnaJ and on ATP hydrolysis by DnaK in the presence of DnaJ and
RepA, with and without Mg chelating agents. Reaction
mixtures were as described under ``Materials and Methods''
for RepA activation and contained the following: no chelators (None),
20 mM citrate (Citrate), 16 mM phosphate
(P
), 0.6 mM pyrophosphate (PP
), or
0.15 mM EDTA (EDTA) as indicated. A, RepA activation
with GrpE (black bars) or without GrpE (gray bars)
was measured as described under ``Materials and Methods.'' B, ATP hydrolysis was measured in RepA activation conditions
containing 0.1 µCi of [
-
P]ATP (4500
Ci/mmol) with GrpE (black bars) or without GrpE (gray
bars) as described under ``Materials and
Methods.''
Since Mg is essential for DnaK ATPase activity, we
thought that it was likely that the interplay of GrpE and
Mg
may effect DnaK ATPase and measured rates of ATP
hydrolysis directly in RepA activation conditions using the protein
concentrations that were optimal for RepA activation. At about 1
µM free Mg
, maintained by citrate,
phosphate, or EDTA, the V
of DnaK ATPase
activity with RepA and DnaJ was lowered 4-6-fold (from 0.70
± 0.05 pmol min
to 0.11-0.17 pmol
min
) and, when maintained by pyrophosphate, 2-fold
(to 0.32 ± 0.03 pmol min
). With these
conditions, GrpE stimulated ATP hydrolysis by DnaK in the presence of
RepA and DnaJ to 0.4-0.8 pmol min
, nearly the V
of DnaK alone (Fig. 1B). The
stimulation of DnaK ATPase by GrpE in RepA activation conditions with 1
µM Mg
correlates nicely with the
stimulation of the RepA activation reaction by GrpE. Moreover, GrpE
lowered the Mg
requirement for ATP hydrolysis by DnaK
in RepA activation conditions from 8 ± 1 µM to 1.5
± 0.5 µM (Fig. 2), similar to its lowering
of the Mg
requirement for RepA
activation(20) . The observations that GrpE stimulates both
DnaK ATPase and RepA activation at 1 µM Mg
and lowers the Mg
requirement for both ATP
hydrolysis and RepA activation suggest that GrpE acts in RepA
activation in vitro to facilitate the utilization of
Mg
by DnaK ATPase and to stimulate ATP hydrolysis.
Figure 2:
Effect of GrpE on the free Mg requirement for ATP hydrolysis in RepA activation. ATP hydrolysis
was measured directly in RepA activation conditions containing 0.1
µCi of [
-
P]ATP (4500 Ci/mmol) and 0.15
mM EDTA, with GrpE (
) or without GrpE
(
).
The
maximal velocity of 0.6 µM DnaK ATPase alone with 20
µM ATP and 10 mM Mg was 0.60
± 0.03 pmol min
at 30 °C, and the
turnover number was 0.05 ± 0.003 min
, similar
to values previously reported(25) . With EDTA as a metal ion
buffer, the Mg
titration curve was sigmoidal, and
half-maximal ATPase required a free Mg
concentration
of 9 ± 1 µM Mg
(Fig. 3).
We obtained a similar Mg
requirement with a 5-fold
higher DnaK concentration and in conditions without EDTA (data not
shown). The sigmoidal shape of the Mg
titration curve
suggests some Mg
-dependent cooperativity in the
function of DnaK ATPase.
Figure 3:
Free Mg requirement for
DnaK ATPase. ATPase assays were performed with 0.6 µM DnaK, 0.2 mM EDTA, and 0.02-10 mM MgCl
, as described under ``Materials and
Methods.''
We observed that GrpE in combination with
DnaJ lowered the Mg concentration required for
half-maximal DnaK ATPase about 10-fold, from 9 ± 1 to 1.5
± 0.5 µM (Fig. 4). In contrast, DnaJ alone
slightly increased the Mg
concentration required for
half-maximal DnaK ATPase to 15 ± 2 µM, when added
in a 2-fold molar excess to DnaK (Fig. 4). This suggests that
the lowering of the Mg
requirement for DnaK ATPase
with DnaJ and GrpE depends specifically on GrpE. GrpE alone lowered the
concentration of Mg
required for DnaK ATPase but only
by about 3-fold, to 3.1 ± 0.3 µM, when added in a
2-fold molar excess to DnaK (Fig. 5). Thus, although GrpE is
responsible for the lowering of the Mg
requirement,
it is more effective in combination with DnaJ than alone.
Figure 4:
Effect of GrpE and DnaJ on DnaK ATPase as
a function of free Mg concentration. ATPase assays
were performed as described under ``Materials and Methods''
with 0.2 mM EDTA and 0.02-10 mM MgCl
and contained 0.6 µM DnaK (
), 0.6 µM DnaK and 1.2 µM DnaJ (
), or 0.6 µM DnaK, 1.2 µM DnaJ, and 1.2 µM GrpE
(
).
Figure 5:
Effect of GrpE on DnaK ATPase as a
function of free Mg concentration. ATPase assays were
performed as described under ``Materials and Methods'' with
0.2 mM EDTA and 0.02-10 mM MgCl
and
contained 0.6 µM DnaK (
), 0.6 µM DnaK
and 0.12 µM GrpE (
), 0.6 µM DnaK and
0.6 µM GrpE (
), or 0.6 µM DnaK and 1.2
µM GrpE (
).
Simultaneously to the lowering of the Mg
requirement, GrpE stimulated the V
of DnaK
ATPase by 3-fold (Fig. 5). With lower molar ratios of GrpE to
DnaK there were correspondingly lesser effects on both the requirement
for Mg
and on the V
of DnaK
ATPase (Fig. 5). DnaJ alone stimulated the V
of the DnaK ATPase about 3-fold, and the combination of GrpE and
DnaJ stimulated the V
about 5-fold, as
previously seen (Fig. 4) (11, 25) .
Interestingly, the joint stimulation of DnaK ATPase by GrpE and DnaJ,
like the stimulation by GrpE alone, was greatest at about 1 µM Mg
, while the stimulation of DnaK ATPase with
DnaJ alone was maximal at about 200 µM of free
Mg
(Fig. 6). This suggests that DnaK ATPase is
optimally stimulated when DnaJ and GrpE interact with DnaK at the same
time, although they may effect different steps of the ATPase reaction.
Figure 6:
The relative stimulation of DnaK ATPase by
DnaJ and GrpE as a function of free Mg concentration.
The ratio of V
/V from Fig. 4and Fig. 5were plotted as a function of free
Mg
concentration. V
is
the velocity of 0.6 µM DnaK ATPase stimulated with 1.2
µM DnaJ (
), 1.2 µM GrpE (
), or
1.2 µM DnaJ and 1.2 µM GrpE (
). V is the velocity of the control 0.6 µM DnaK
ATPase.
Figure 7:
Effect of GrpE on the ATP requirement for
DnaK ATPase at 1 µM free Mg. ATPase
assays were performed as described under ``Materials and
Methods'' with 0.15 mM EDTA and 0.1 mM MgCl
and contained 0.6 µM DnaK (
) or
0.6 µM DnaK and 1.2 µM GrpE
(
).
Without Mg GrpE increased the K
for ATP binding by DnaK
by 100-fold, from about 0.5 µM to at least 100
µM, as measured directly by equilibrium dialysis
experiments (Fig. 8). Therefore, GrpE lowers the affinity of
DnaK for nucleotide more dramatically without than with
Mg
.
Figure 8:
GrpE effect on ATP binding by DnaK without
Mg. DnaK (
) or DnaK with equimolar amounts of
GrpE (
) were dialyzed for 42 h at 4 °C with a concentration
series of 0.004 nM to 100 µM [
-
P]ATP as described under
``Materials and Methods.'' The free Mg
concentration was controlled at about 10 nM by 20 mM EDTA. The amount of bound and free
[
-
P]ATP was determined by liquid
scintillation counting.
The stimulation of the nucleotide exchange from DnaK was proposed to be the major function of GrpE, which directly or indirectly controls the binding and release of polypeptide substrates by DnaK(11, 26, 30) . Despite its importance, little is known about the mechanism by which the association of GrpE with DnaK releases the nucleotide and by which ATP can rebind to the DnaK-GrpE complex.
Our search to explain the GrpE requirement for
RepA activation lead us to a surprising finding that DnaK ATPase in the
presence of GrpE requires lower Mg concentration and
simultaneously higher ATP concentration. The additional observation
that GrpE lowers the affinity of DnaK for ATP much more dramatically
without than with Mg
suggests the possibility that
nucleotide release requires not only the lowering of the affinity of
DnaK for nucleotide but also the weakening of the
Mg
-nucleotide interactions. This may be accomplished
by a rearrangement of the negatively charged amino acid residues
ligating Mg
in the active center of DnaK ATPase such
that Mg
would be at least transiently bound by DnaK
with an increased affinity. The crystallographic analysis of the
structure of the ATPase fragment of bovine brain Hsc70 supports the
possibility that rearrangements of residues Asp-10 and Glu-175 may
result in strengthening the ligation of
Mg
(27) . Moreover, it has been shown that
GrpE efficiently releases ATP from DnaK prior to ATP
hydrolysis(11) .
We have demonstrated that GrpE lowers the
Mg requirement for DnaK ATPase both with RepA
activation conditions and with DnaK ATPase conditions. This suggests
that the increased DnaK affinity for Mg
observed with
GrpE is important for hydrolysis of ATP bound to the DnaK-GrpE complex.
This observation also brings into question whether or not the
stimulation of DnaK ATPase by GrpE is due solely to its nucleotide
exchange function. It is possible that the effect of GrpE on the
ligation of Mg
contributes to the stimulation of DnaK
ATPase, especially in conditions with high concentrations of ATP when
the inhibition by ADP and P
products is negligible.
The
control of GrpE over DnaK amino acid residues ligating Mg may also be important for discrimination against the binding of
other divalent metal ions to the active site of DnaK ATPase. The
precise position and electrostatic environment of Mg
in the active center of Hsc70 ATPase was suggested to be critical
for the ATPase activity, based on the observation that all the residues
ligating Mg
are highly conserved and cannot be
changed without affecting ATPase activity(27, 28) .
Such a high conservation of the Mg
ligands in the
ATPase active site suggests that Hsp70 may be very selective in its
requirement for Mg
and sensitive for any
rearrangements of the Mg
environment. Contrary to
that, Hsp70s are functional even in the presence of heavy metals that
stimulate the heat shock response. The GrpE-dependent discrimination of
other metal ions from binding to DnaK is an intriguing hypothesis that
provides a link between the specific requirement for GrpE observed in
the in vitro RepA activation with DnaK and DnaJ to utilize
Mg
for DnaK ATPase at low Mg
concentration and the requirement for GrpE in vivo,
likely to facilitate the utilization of Mg
in the
presence of other metal ions competing for binding to DnaK.