(Received for publication, October 10, 1995; and in revised form, January 8, 1996)
From the
Applying stopped-flow fluorescence spectroscopy for measuring
conformational changes of the DnaK molecular chaperone (bacterial Hsp70
homologue) and its binding to target peptide, we found that after ATP
hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which
possesses limited affinity for peptide substrates and the GrpE
cochaperone but efficiently binds the DnaJ chaperone. In the presence
of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted
back to the DnaK conformation, and the resulting DnaJDnaK(ADP)
complex binds to peptide substrates more tightly. Formation of the
DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The
presence of GrpE and ATP hydrolysis promotes the fast release of the
peptide substrate from the chaperone complex and converts DnaK to the
DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and
GrpE, the binding-release cycle of DnaK is stoichiometrically coupled
to the adenosine triphosphatase activity of DnaK.
The Hsp70 molecular chaperone (Ellis, 1993), by transient binding to different denatured (Gragerov et al., 1994; Fourie et al., 1994) or native polypeptides (Wawrzynów and Zylicz, 1995), makes these substrates suitable for transport (Hendrick et al., 1993) and proteolysis (Sherman and Goldberg, 1992), sequesters unfolded peptides thus promoting their correct folding (Szabo et al., 1994), and protects or reactivates heat labile enzymes (Skowyra et al., 1990; Schroder et al., 1993; Ziemienowicz et al., 1993, 1995). Hsp70 also disassembles the clathrin-coated vesicles during receptor-mediated endocythosis (Schlossman et al., 1984) and activates the formation of protein complexes involved in the initiation of transcription (Blaszczak et al., 1995), DNA replication (Alfano and McMacken, 1989; Zylicz et al., 1989; Wickner et al., 1992), or the assembly of the glucocorticoid receptor complex (Hutchison et al., 1992).
The Hsp70 cooperates in these reactions with two other cochaperones, Hsp40 (the bacterial DnaJ homologue; Zylicz et al., 1985; Silver and Way, 1993) and Hsp24 (the bacterial GrpE homologue; Zylicz et al., 1987; Bolliger et al., 1994; Laloraya et al., 1994). The Escherichia coli DnaK/DnaJ/GrpE chaperone system can therefore serve as a paradigm for the analysis of the Hsp70 chaperone machine.
The bacterial DnaK protein, like its eukaryotic Hsp70
homologue, possesses a weak K-dependent ATPase
activity (Zylicz et al., 1983; O'Brien and McKay, 1995),
which is stimulated 2-20-fold in the presence of peptide
substrates (Zylicz et al., 1983; Jordan and McMacken, 1995).
In the absence of peptide substrates, under steady-state conditions,
the ATPase of DnaK is highly stimulated in the joint presence of the
GrpE and DnaJ cochaperones (Liberek et al., 1991a; Jordan and
McMacken, 1995; McCarty et al., 1995). The situation is
different in the presence of a protein substrate. In this case, DnaJ
does not affect the peptide-dependent ATPase activity of DnaK, but the
presence of GrpE vigorously stimulates this activity (Jordan and
McMacken, 1995).
In this paper using methodology that allowed us to follow the real-time kinetics of DnaK/DnaJ/GrpE molecular chaperone action, we were able to show that the presence of DnaJ induces such DnaK conformations that the stable complex between peptide substrates and DnaK is formed. GrpE and ATP hydrolysis are required for the recycling of DnaK from this complex, thus allowing multiple substrate binding-release cycles of DnaK chaperone action.
Figure 1:
Intrinsic fluorescence of the
DnaK's tryptophan. The fluorescence of DnaK (2 µM)
was measured as described under ``Experimental Procedures''
in the absence or presence of 1 mM ATP or 1 mM ADP
supplemented with 1 mM K/HPO. Similar results were
obtained when ADP and K/HPO
were added after the first
turnover of DnaK ATPase (result not shown).
In this paper the conformation occurring after incubation of DnaK
with ATP is referred as DnaK*. To monitor such conformational changes
of DnaK we used the stopped-flow technique and measured the kinetics of
the decrease of DnaK's tryptophan fluorescence after rapidly
mixing DnaK with ATP (Fig. 2A). The rate of this
reaction (t, time of 50% amplitude change t
= 30.3 ± 0.1 s; assuming pseudo
first-order kinetics k
= 0.023 ±
0.0016 s
) does not change with an increase in ATP
concentration (Fig. 2A and result not shown). The
substitution of ATP by either ADP, ADP + P
, or AMP-PCP (
)did not result in any changes of the tryptophan
fluorescence of DnaK ( Fig. 1and Fig. 2A and
results not shown), suggesting that the ATP hydrolysis, rather than
nucleotide binding, is a major factor influencing change of the
tryptophan fluorescence of DnaK. According to the previously published
results, one molecule of ATP is hydrolyzed by DnaK every 10-15
min (Zylicz et al., 1983; Liberek et al., 1991a;
Palleros et al., 1993; Schmid et al., 1994; Jordan
and McMacken, 1995; McCarty et al., 1995), whereas the
observed DnaK conformational changes occur much more rapidly (t
= 30.3 s). To solve this discrepancy,
we showed that the purified monomeric form of DnaK (nucleotide-free),
used in these experiments, hydrolyzed ATP molecule at a much faster
rate (k
= 0.025 ± 0.005
s
; see Fig. 2B). However, after a
single round of ATP hydrolysis, the rate of the reaction decreased and
reached the previously estimated steady-state value (Fig. 2B). This burst of ATP hydrolysis could not be
detected previously, because in those cases at least 10-fold lower
concentrations of DnaK (which was a mixture of monomeric and multimeric
forms) were used.
Figure 2:
Kinetics of conformational changes of
DnaK during the ATP hydrolysis. A, real time kinetics of
conformational changes of DnaK were measured directly by following the
relative fluorescence of the single tryptophan residue located at the
N-terminal domain of the DnaK chaperone. The ATP (or ADP) was injected
(final concentration, 100 µM) to a highly purified,
monomeric DnaK (5 µM), and the change of
tryptophan's fluorescence was monitored using stopped-flow
spectrofluorimeter as described under ``Experimental
Procedures.'' The insert represents the same data plotted in the
scale from 0 to 5 s to show that fast reaction of ADP binding does not
change the DnaK's tryptophan fluorescence. B, ATP
hydrolysis catalyzed by DnaK. DnaK protein (5 µM) and ATP
(100 µM) was incubated with () or without 500
µM K/HPO
(
). At the desired time, the
reaction was stopped using two volumes of 8 M urea, and the
amount of ADP formed during the hydrolysis reaction was estimated as
described by Gao et al. (1993). The insert represents the data
obtained following longer times of incubation. The DnaK monomer and
buffer conditions were as described under ``Experimental
Procedures.''
Results presented in Fig. 1and Fig. 2reveal that the hydrolysis of ATP occurs with a pre-steady
state burst and that the dissociation of P and ADP is
rate-determining. Supporting this, the burst of product formation is
detected in the absence of exogenous P
but inhibited in the
presence of P
(Fig. 2B). The diagram of the
DnaK ATPase reaction is proposed in Fig. 3A. The binding of
ATP to the DnaK is a fast reaction (t
< 0.5 s; K
= 35 nM) (Montgomery et
al., 1993). This induces conformational changes of DnaK (Palleros et al., 1992; Banecki et al., 1992) that cannot be
monitored by intrinsic fluorescence of the tryptophan ( Fig. 1and Fig. 2and result not shown). The binding of
ATP to DnaK (k
; Fig. 3A) is not
inhibited by the presence of exogenous P
(result not
shown). After the ATP hydrolysis (k
; Fig. 3A) both products (ADP and P
), which
are present in the active site of DnaK ATPase dissociate from DnaK
complex at different rates. After the conformational change of DnaK
(conversion to DnaK* form), the P
product is released (k
; Fig. 3A). These conformational
changes are inhibited by the presence of exogenous P
(Fig. 4A). Interestingly, the preincubation of
DnaK with both ADP and P
does not change the tryptophan
fluorescence of DnaK (Fig. 1), suggesting that the DnaK*(ADP)
conformation could by reached only after ATP hydrolysis.
Figure 3:
A, the diagram of the DnaK ATPase
reaction. B, DnaJ- and GrpE-dependent conformational changes
of DnaK protein. See ``Results'' for details. Please note
that (DnaJDnaK
GrpE) is a transient
intermediate.
Figure 4:
Kinetics of conformational changes of DnaK
in the presence of the DnaJ cochaperone. A, DnaK (5
µM) was rapidly mixed with ATP (500 µM) in
the presence or absence of 500 µM K/HPO. B, DnaK was rapidly mixed with ATP and after 0, 10, 50 or 150
s DnaJ (10 µM) was injected. The change of DnaK's
tryptophan fluorescence was recorded using the stopped-flow
spectrofluorimeter as described under ``Experimental
Procedures.'' C, the experiment was performed as in panel B, except after 150 s preincubation of DnaK with ATP,
DnaJ was added with 500 µM K/HPO
.
These results suggest that the
presence of DnaJ induces the DnaK conformational changes such that
P (and to some extend also ADP) can dissociate from the
complex. This idea can be supported by the fact that the presence of
DnaJ partially releases the
ADP fluorescence analogue from the
DnaK
ADP complex formed after incubation of
ATP with
DnaK. However, when DnaJ in this reaction is substituted by the
GrpE-nucleotide exchange factor, the release of
ADP from the
complex is much more efficient. (
)
To monitor
the conformational changes of DnaK induced by the presence of GrpE, we
mixed DnaK,ATP with GrpE. In the case where DnaK was not converted to
DnaK*(ADP) form (Fig. 5A, delay 0 s), a fast decrease of
DnaK's tryptophan fluorescence was observed (t = 0.24 ± 0.05 s; Fig. 5B). In
contrast, in the absence of GrpE, the reaction was much slower (Fig. 5A, t
= 30.3 s).
The presence of GrpE appears to accelerate conformational changes of
DnaK induced after the ATP hydrolysis. In the same time, GrpE (in the
absence of protein substrates) does not accelerate the rate of ATP
hydrolysis (Jordan and McMacken, 1995), suggesting that GrpE works
mostly as a nucleotide exchange factor. An 8-fold decrease in GrpE
concentration did not significantly affect this reaction (result not
shown), suggesting that GrpE can be recycled. Probably, when all three
components are mixed together (DnaK, GrpE, and ATP), the reaction
proceeds with additional intermediatory steps. First GrpE binds to
DnaK, and then, upon ATP hydrolysis, DnaK is converted to the
DnaK*(ADP) form resulting in the release of GrpE. However, when the
DnaK protein was first preincubated with ATP, DnaK's tryptophan
fluorescence was less sensitive to the presence of GrpE (Fig. 5B, delay 150 s). These results suggest that
DnaK*(ADP) conformation possesses limited affinity to GrpE cochaperone.
Figure 5:
Conformational changes of DnaK in the
presence of GrpE or both GrpE and DnaJ. A, DnaK (5
µM) was rapidly mixed with ATP (500 µM), and
fluorescence of DnaK's tryptophan was monitored as described in Fig. 1. B, DnaK (5 µM) was mixed with ATP
(500 µM) and after 0, 50, or 150 s GrpE (20
µM) was injected. C, DnaK (5 µM) was
rapidly mixed with a solution containing DnaJ (10 µM) and
ATP (500 µM) and after preincubation for 150 s, GrpE (20
µM) was added: stopped-flow trace
[DnaK+(DnaJ+ATP)] GrpE. The experiment was
repeated but ATP was substituted by 500 µM of ADP:
stopped-flow trace [DnaK+(DnaJ+ADP)]
GrpE.
A different situation is observed when DnaJ is added to this
reaction (Fig. 5C). The preincubation of DnaK, ATP, and
DnaJ resulted in the formation of the DnaJDnaK(ADP) complex (Fig. 3, reactions 1). In the continuous presence of ATP, the
addition of GrpE to this complex triggered the very fast conformational
changes of DnaK (t
= 0.18 ± 0.05
s) to the DnaK*(ADP) form (Fig. 5C). We suggest that
GrpE releases ADP from the DnaJ
DnaK(ADP) complex first (Fig. 3B, reaction 2; see also Liberek et al. (1991a)) and then an intermediate
[DnaJ
DnaK
GrpE] complex is formed. The formation
of this transient intermediate is suggested also by the fact that the
simultaneous presence of GrpE and DnaJ stimulates DnaK's ATPase
activity from 50- up to 180-fold (Liberek et al., 1991a;
McCarty et al., 1995). Subsequently after the ATP hydrolysis (Fig. 3B, reaction 3), DnaK is converted to the
DnaK*(ADP) form and GrpE is released from the chaperone complex. In the
control experiment, we showed that ATP
S could trigger the release
of DnaJ from DnaK complex (Wawrzynów and Zylicz,
1995). This facts suggest that DnaJ could leave chaperone complex
before GrpE dissociation.
As reported previously by Schmid et al.(1994), in the
absence of ATP, DnaK binds efficiently to ap1 peptide (Fig. 6A). To monitor the ATP-dependent release of DnaK from
its substrate complex, we preincubated DnaK with ap1 peptide and then
added ATP. When all three factors were rapidly mixed, the increase of
DnaK's binding to ap1 was observed (Fig. 6B, 0 s
delay). When DnaK and ap1 were incubated for 100 s before the addition
of ATP, we observed the release of DnaK from its complex with ap1
peptide (t = 17 ± 0.1 s; Fig. 6B). The increase in the rate of this reaction (k
= 0.042 ± 0.002
s
) comparable wiht the rate of conformational
changes of DnaK induced after ATP hydrolysis, observed in the absence
of the peptide substrate (Fig. 2A; k
= 0.027 s
) could be due to the peptide
dependent-stimulation of DnaK's ATPase (Jordan and McMacken,
1995).
Figure 6:
The
binding of DnaK to the ap1 peptide substrate in the presence or absence
of ATP, DnaJ and GrpE cochaperones. A, DnaK (5
µM) was preincubated for 100 s before acrylodan-modified
ap1 peptide (0.5 µM) was injected: stopped-flow trace
DnaK ap1. The change of acrylodan fluorescence after binding of
DnaK to ap1 peptide was monitored using stopped-flow spectrofluorimeter
as described under ``Experimental Procedures.'' B,
DnaK (5 µM) was rapidly mixed with the ap1 peptide (0.5
µM) and after 0, 10, or 100 s of preincubation, ATP (500
µM) was injected. C, effect of the DnaJ chaperone
on binding of DnaK to the ap1 peptide substrate. DnaK (5
µM) was rapidly mixed with a solution containing ATP (500
µM) and after 100 s preincubation the ap1 peptide (0.5
µM) was added: stopped-flow trace (DnaK+ATP)
ap1. In the second experiment the DnaK (5 µM), ATP (500
µM), and ap1 (0.5 µM) were incubated for 100
s before DnaJ (10 µM) was injected: stopped-flow trace
[DnaK+(ap1+ATP)]
DnaJ. D, effect of
the GrpE on the stability of the DnaJ[ap1-DnaK(ADP)] complex.
DnaK (5 µM) was rapidly mixed with a solution containing
DnaJ (10 µM), ap1 (0.5 µM), and ATP (500
µM). Following 0, 10, or 200 s of incubation, GrpE was
added and the fluorescence of the acrylodan-bound peptide was
recorded.
In the situation when DnaK is converted to the DnaK*(ADP) conformation, the binding to the peptide substrate was faster, but the amplitude of the fluorescence changes was much smaller (Fig. 6C, delay 100 s) than in the absence of ATP (Fig. 6A). These observations suggest that the presence of hydrolyzable ATP simple shifts the equilibrium of DnaK/substrate reaction in favor of dissociation.
The presence of DnaJ changes the
kinetics of this reaction. In the presence of ATP, DnaJ substantially
stabilizes the ap1DnaK complex (Fig. 6C). In this
case DnaJ probably inhibits the dissociation of ap1 from ap1
DnaK
complex. An increase in substrate-DnaK complex stability in the
presence of DnaJ was previously noted using different experimental
approaches (for review, see Wawrzynów et
al.(1995)).
To test the kinetics of GrpE action,
we preincubated DnaK, DnaJ, and ATP with ap1 peptide and then injected
GrpE (Fig. 6D). After a short preincubation of
0-10 s, almost no effect was observed (Fig. 6D,
delay 0 and 10 s). As shown in Fig. 6C, at least
100-200 s is required for
DnaJ[ap1
DnaK(ADP)] complex formation. When the
preincubation was prolonged to 100 or 200 s, the addition of GrpE (in
the presence of ATP) triggered an efficient and fast release of the
substrate (Fig. 6D, delay 200 s). As shown in Fig. 5, in this case DnaK is converted back to the DnaK*(ADP)
conformation. The GrpE-dependent disassociation of DnaK from ap1
complex is fast (t
= 0.3 ± 0.05
s), suggesting that the action of two cochaperones, DnaJ and GrpE, is
coupled to ATP hydrolysis and probably accomplished through a
DnaJ
(ap1
DnaK
GrpE) intermediate.
According to the data presented in this paper, the reaction
of the DnaK-dependent ATP hydrolysis occurs with a pre-steady state
burst, when initiated in the presence of nucleotide-free monomeric DnaK
protein. The products of this reaction (ADP + P)
suppress the turnover of the subsequent hydrolysis reactions. After the
first round of ATP hydrolysis, DnaK is converted to the DnaK*(ADP)
conformation. Three facts suggest that this DnaK conformational change
occurs not during the binding of ATP to DnaK but after the ATP
hydrolysis.
1) The rate of DnaK's conformational change is not influenced by the increase in ATP concentration (this paper).
2) The
exogenous P inhibits both ATP hydrolysis and DnaK
conformational change but does not influence the binding of ATP to DnaK
(this paper).
3) The absence of K inhibits
DnaK's ATPase activity and conformational change but not the
binding of ATP to DnaK (Palleros et al., 1993).
Assuming a high concentration of ATP and the presence of both cochaperones (DnaJ and GrpE), most of DnaK in vivo should then be in the DnaK*(ADP) form. This suggests that the entry (Fig. 3A) to the DnaK/DnaJ/GrpE cycle is significant only in an in vitro situation where DnaK-nucleotide-free form could be reached.
The
scheme in Fig. 7summarizes the results presented in this paper.
In the presence of DnaJ, the DnaK*(ADP) form is converted to the DnaK
conformation (Fig. 7, reaction 1), and the DnaJDnaK(ADP)
complex binds to its peptide substrate tightly (Fig. 7, reaction
2). In the control experiments, we have shown that the DnaK*(ADP) form
binds protein substrates in a very fast reaction but that in the
absence of DnaJ, DnaK rapidly dissociates from its substrate-DnaK
complex. The DnaJ-dependent transition of DnaK*(ADP) to DnaK
conformation is not inhibited by the exogenous P
,
suggesting that the ATP hydrolysis is not required for this transition
reaction. The formation of the active DnaJ
DnaK(ADP) complex is
limited by the DnaJ concentration. In vivo in E. coli bacterial cells there is at least 10-fold less DnaJ than DnaK
(Bardwell et al., 1986). In the cell, probably most of DnaK
protein is in the DnaK*(ADP) form, which binds and releases protein
substrates very fast. Only this DnaK, which is activated by DnaJ (or
these substrates that are ``tagged'' by DnaJ) will form a
stable substrate-DnaK complex.
Figure 7: Model of the DnaK/DnaJ/GrpE chaperone machine interaction with a peptide substrate (ap1). See ``Discussion'' for details.
The ap1DnaK(ADP) complex, which
is probably transiently bound to DnaJ, is being formed after the
DnaJ-dependent activation of DnaK*(ADP) (Fig. 6, reaction 2).
This is a rate-limiting step in DnaK/DnaJ/GrpE cycle. The rate of this
reaction is dependent on the peptide concentration and its amino acid
sequence or/and peptide conformation. In this case, the affinity of the
peptide to DnaJ is important (Wawrzynów and
Zylicz, 1995; Wawrzynów et al., 1995).
The presence of ADP stabilizes the
DnaJ
[ap1
DnaK(ADP)] complex
(Wawrzynów et al., 1995). The mechanism
of ADP-dependent stabilization of DnaK-substrate complex should be
significant in vivo. During stress conditions, the ATP
concentration dramatically drops down and ADP concentration increases
(Findly et al., 1983). Such situations can
``freeze'' Hsp70-substrate complexes helping the Hsp70 to
protect other protein from inactivation.
The GrpE, after the ATP
hydrolysis, triggers the fast release of DnaK from its protein
substrate and converts DnaK back to the DnaK*(ADP) form (Fig. 7,
reaction 3). The GrpE is known as a nucleotide exchange factor, which
after release of ADP from DnaK complex could lead to the formation of
[substrateDnaK
GrpE] intermediate (Osipiuk et
al., 1993). The GrpE also, in the presence of protein substrate
stimulates DnaK's ATPase activity (Jordan and McMacken, 1995).
Our data do not exclude the existence of any additional intermediate
complexes during the binding of ATP and ATP hydrolysis. DnaJ probably
dissociates first from the substrate-chaperone complex.
The
experiments described in this paper were performed using the ap1
peptide, which does not directly interact with the DnaJ protein (result
not shown). In the case where the protein substrate binds to DnaJ
(P, denatured luciferase, RepA,
), the DnaJ
chaperone in addition to the DnaK-substrate stabilization effect, can
also contribute in delivery of the protein substrate to DnaK (Liberek et al., 1990; Wickner et al., 1992; Gamer et
al., 1992; Langer et al., 1992; Szabo et al.,
1994; Wawrzynów et al., 1995). In these
cases the substrate-DnaJ complex will directly bind to DnaK*(ADP) (Fig. 7, reaction 2`). DnaJ remaining tightly bound to the
substrate can promote several ATP-dependent cycles of DnaK
conformational changes (Szabo et al., 1994). Such multiple
binding and release steps of DnaK from the DnaJ-substrate complex can
induce conformational changes to the protein substrates, thus leading
to the activation of some proteins or reactivation of heat-inactivated
enzymes. Using FTIR spectroscopy we found that
C-labeled
DnaK protein indeed induces (or freezes) conformational changes to the
C-labeled RepA protein substrate.
Recently, we were able to show, using two independent experimental approaches (size high performance liquid chromatography and the enzyme-linked immunosorbent assay), that the presence of ATP and DnaJ stabilizes the DnaK bound not only to the denatured but also to the native polypeptide substrates (Wawrzynów et al., 1995). Therefore, the results presented in this paper represent general phenomena, which describe the role of DnaJ and the GrpE cochaperones in DnaK-substrate complex formation and dissociation.