(Received for publication, February 10, 1995; and in revised form, June 6, 1995)
From the
Using the native proteins P,
O,
,
and RepA, as well as permanently unfolded
-carboxymethylated
lactalbumin, we show that DnaK and DnaJ molecular chaperones possess
differential affinity toward these protein substrates. In this paper we
present evidence that the DnaK protein binds not only to short
hydrophobic peptides, which are in an extended conformation, but also
efficiently recognizes large native proteins (RepA,
P). The best
substrate for either the DnaK or DnaJ chaperone is the native P1 coded
replication RepA protein. The native
transcription
factor binds more efficiently to DnaJ than to DnaK, whereas unfolded
-carboxymethylated lactalbumin or native
P binds more
efficiently to DnaK than to the DnaJ molecular chaperone. The presence
of nucleotides does not change the DnaJ affinity to any of the tested
protein substrates. In the case of DnaK, the presence of ATP inhibits,
while a nonhydrolyzable ATP analogues markedly stimulates the binding
of DnaK to all of these various protein substrates. ADP has no effect
on these reactions. In contrast to substrate protein binding, DnaK
binds to the DnaJ chaperone protein in a radically different manner,
namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits
the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein
lacking 94 amino acids from its C-terminal domain, which still
possesses the ATPase activity and forms a transient complex with
protein substrates, does not interact with DnaJ protein. We conclude
that the DnaK-ADP form, derived from ATP hydrolysis, possesses low
affinity to the protein substrates but can efficiently interact with
DnaJ molecular chaperone.
The highly conserved and ubiquitous 70-kDa heat shock proteins
(Hsp70), or the prokaryotic DnaK homologues, function as molecular
chaperones in a variety of cellular processes (reviewed by Gething and
Sambrook(1992), Hendrick and Hartl(1993), Georgopoulos et
al.(1994), and Craig et al.(1994)). For example, Hsp70
chaperones have been shown to be involved in the binding of nascent
polypeptides (Beckman et al., 1990), protein transport
(Neupert et al., 1990; Scherer et al., 1990; Wild et al., 1992; Hendrick et al., 1993; Brodsky et
al., 1993), the disassembly of clathrin-coated vesicles (Chappell et al., 1986; Schmid and Rothman, 1985a, 1985b; Schmid et
al., 1985; Prasad et al., 1994), the activation of the
p53 anti-oncogene to bind to specific DNA sequences (Hupp et
al., 1992), the DNA replication of bacteriophages (Zylicz,
1993) and P1 (Wickner et al., 1992), and the autoregulation of
the heat shock response (Yura et al., 1993), as well as in
vivo proteolysis (Sherman and Goldberg, 1992). The Hsp70
chaperones can function in these diverse cellular processes because
they exhibit promiscuous substrate binding properties (BlondElguindi et al., 1993a; Gragerov et al., 1994). For instance,
Hsp70 generally recognizes nascent polypeptides chains, peptides in the
extended conformation (Flynn et al., 1991; Landry et
al., 1992), and unfolded proteins (bovine pancreatic trypsin
inhibitor; RNA polymerase, CMLA, (
)rhodanese; for a review,
see Hendrick and Hartl(1993)). Through binding to the hydrophobic
unfolded regions of proteins, the Hsp70 chaperones not only prevent
protein aggregation, but can even disassociate protein aggregates, thus
facilitate protein folding/assembly (Pelham, 1986; Skowyra et
al., 1990; Schroder et al., 1993; Ziemienowicz et
al., 1993). (
)In addition, Hsp70 can bind and activate
native proteins, which necessitate conformational changes for their
activation, i.e.
P,
O (Liberek et al.,
1990), RepA (this paper), p53 (Hupp et al., 1992), and
(Liberek et al., 1992; Gamer et
al., 1992; Liberek and Georgopoulos, 1993).
Crucial to Hsp70's mode of action is its ability to bind and hydrolyze ATP (Zylicz et al., 1983; Welch and Feramisco, 1985). Hsp70 has a relatively high affinity for ATP and an extremely weak ATPase activity (for a detailed review see, McKay et al.(1994) and Hightower et al.(1994)), which can be stimulated up to 20-fold by peptides or certain protein substrates (Hightower et al., 1994; Blond-Elguindi et al., 1993b; Buchberger et al., 1994). The binding and hydrolysis of ATP results in conformational changes to the various Hsp70 family members (Liberek et al., 1991b; Palleros et al., 1992; Banecki et al., 1992). Recently it was suggested (under conditions where DnaJ was not present) that the binding, but not ATP hydrolysis, is required for the disassociation of Hsp70 from its substrate (Palleros et al., 1993; Schmid et al., 1994).
Most models addressing the question of how Hsp70 functions in protein transport or folding assume that Hsp70 bound to ADP (Hsp70-ADP) is the form exhibiting the highest affinity toward the substrate and ATP hydrolysis is required only for the regeneration of such a form (for review, see Craig et al.(1994)). This conclusion was based on the findings that Hsp70 family members form a stable complex with various substrates in the presence of ADP but not ATP (Palleros et al., 1991). However, recently stopped-flow measurements have revealed that the DnaK chaperone exhibits a faster on rate for peptides when bound to ATP than ADP, but it exhibits an even faster off rate when bound to ATP. Thus, overall the DnaK-substrate complex is less stable in the presence of ATP than ADP (Schmid et al., 1994).
In this paper we show that the product of ATP hydrolysis, namely the DnaK-ADP form possesses limited affinity to the protein substrates but binds efficiently to the DnaJ protein.
Anti-DnaK and anti-DnaJ rabbit sera were generated as described by Zylicz and Georgopoulos(1984). Protein concentrations were estimated by the Bio-Rad assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie Blue staining.
Figure 1:
Interaction of the DnaK and DnaJ
molecular chaperones with various protein substrates. Panels A and C, the various protein substrates, namely: ,
RepA;
,
P;
,
O; ▴,
;
, CMLA; and ▪, BSA, were preincubated (0.5 µg/well) in
ELISA plate wells in PBS buffer as described under ``Materials and
Methods.'' Following the washing procedure with PBS/BSA and A/BSA
buffers, an increasing amount of either DnaK (panel A) or DnaJ (panel C) in buffer A/BSA was added and the volume adjusted to
60 µl. After a 30-min incubation at room temperature, the proteins
were cross-linked with glutaraldehyde as described under
``Materials and Methods.'' The amount of DnaK (panel
A) or DnaJ (panel C) bound to the various protein
substrates was detected using either anti-DnaK or anti-DnaJ serum,
respectively, as described under ``Materials and Methods.'' Panels B and D, the CMLA (0.5 µg/well) was
preincubated on ELISA plate, as described above. After washing with
PBS/BSA and A/BSA buffer, increasing amounts of protein substrates:
, RepA;
,
P;.
,
O; ▴,
; and
, CMLA were preincubated for 15 min at
30 °C with 80 µg of DnaK (panel B) and 40 µg of
DnaJ (panel D) in A/BSA buffer was added and incubation
proceeded for an additional 15 min. After cross-linking with
glutaraldehyde and washing, the amount of DnaK (panels A and B) and DnaJ (panels C and D) bound to CMLA
was detected using either anti-DnaK or anti-DnaJ serum,
respectively.
Previously we had shown that DnaK
possesses a weak ATPase activity resulting in the hydrolysis of 1
molecule of ATP every 10 min (Zylicz et al., 1983; Liberek et al., 1991a). In addition, it was shown that ATP causes the
release of DnaK from P,
O, unfolded bovine pancreatic trypsin
inhibitor, and RepA, albeit inefficiently (Fig. 2) (Liberek et al., 1990, 1991b). In the case of
and
CMLA, which possess a lower (without nucleotides) affinity toward DnaK,
the ATP-dependent dissociation of DnaK from these substrates was more
pronounced (Fig. 2). These results suggest that the presence of
ATP simply shifts the equilibrium of the DnaK/substrate reaction in
favor of dissociation. Under these conditions, ADP has only a modest
effect on the binding of DnaK to some of its protein substrates (Fig. 2). Surprisingly, the nonhydrolyzable ATP analogue
(AMP-PCP) efficiently stimulates the binding of all protein substrates
to DnaK (Fig. 2). The stimulatory effect of AMP-PCP was even
more pronounced for the
and CMLA substrates (Fig. 2). In fact, in the presence of AMP-PCP, DnaK exhibits a
similar binding property for all tested protein substrates (Fig. 2). Recently, Palleros et al.(1993) reported that
the ATP
S nonhydrolyzable analogue causes the partial release of
CMLA from its complex with DnaK. To address this apparent discrepancy
we tested two other nonhydrolyzable ATP analogues for their effect on
substrate binding to DnaK. All three ATP analogues tested, namely,
AMP-PCP, AMP-PNP, and ATP
S, stimulated binding of DnaK to
P
or CMLA to the same extend (Fig. 3). Nevertheless, because
during the course of these experiments we found that ATP
S was
unstable we decided to exclusively use AMP-PCP.
Figure 2:
Nucleotides modulate the binding of the
DnaK chaperone to its various substrates. The various protein
substrates (indicated in left top corner of each panel) were
preincubated (0.5 µg/well) in ELISA plate wells as described under
``Materials and Methods.'' After the blocking and washing
procedures, an increasing amount of DnaK chaperone was added in buffer
A/BSA supplemented with the indicated nucleotide (1 mM):
, ATP;
, ADP;
, AMP-PCP; and
, without
nucleotide. After incubation for 30 min at room temperature and
cross-linking with glutaraldehyde, the amount of the DnaK chaperone
bound to various protein substrates was estimated using an anti-DnaK
serum as described under ``Materials and
Methods.''
Figure 3:
Effect of nonhydrolyzable ATP analogues on
the binding of the DnaK chaperone to either P or CMLA.
P (0.5
µg/well, panel A) or CMLA (0.5 µg/well, panel
B) proteins were bound to ELISA plate wells as described under
``Materials and Methods.'' The binding of increasing amounts
of the DnaK chaperone in the presence (1 mM) of:
,
AMP-PCP; ▪, ATP
S; ▴, AMP-PNP; or
, without
nucleotides, was measured as described in the legend of Fig. 2and detailed under ``Materials and
Methods.''
Next, we examined
the influence of nucleotides on the stability of the DnaK-substrate
complexes. DnaK was preincubated with P or CMLA in the presence of
AMP-PCP for 30 min at room temperature (similar results were obtained
when no nucleotides were used), then AMP-PCP and unbound protein were
removed. Following this, fresh buffer with the indicated nucleotides
were added and the reaction was stopped at various times by
cross-linking with glutaraldehyde. The DnaK-
P or DnaK-CMLA
complexes were relatively stable in the absence of nucleotides or in
the presence of ADP but in the presence of ATP nearly all DnaK bound to
either CMLA or
P was released within 5 min (results not shown).
To further substantiate our results obtained from the ELISA
experiments, we also performed size exclusion chromatography using a
Superdex HR 200 column (Pharmacia). When P was incubated with DnaK
(2:1 molar ratio, respectively) a peak was detected which corresponds
with a dimer of
P bound to a monomer of DnaK (Fig. 4). This
protein complex was previously identified by us (Osipiuk et
al., 1993). The presence of ATP in the premixture did not disrupt
the DnaK-
P complex (results not shown). However, when ATP was
present both in the premixture and in the column buffer, the
DnaK-
P complex was disrupted (Fig. 4). This result suggests
that when ATP is not present in the column buffer, the
P-DnaK
complex reforms due to the high affinity of DnaK to
P. In contrast
to
P, the presence of ATP in the premixture only was sufficient to
disrupt the DnaK-CMLA complex (Fig. 4), again suggesting that
DnaK forms a weaker complex with CMLA than
P (Fig. 1A). The presence of ADP had no detectable effect
on the DnaK-
P or DnaK-CMLA complexes (Fig. 4). In good
agreement with the reported ELISA-type of experiments (Fig. 2)
is the observation that the presence of AMP-PCP in the premixture
stimulates binding of DnaK to
P (Fig. 4). An even more
pronounced stimulatory effect of AMP-PCP was found for the CMLA
protein substrate than for
P (Fig. 4). Interestingly, when
AMP-PCP or ATP
S were also present in the column buffer, a lower
amount of CMLA was found in a complex with DnaK, suggesting that
nonhydrolyzable ATP analogues could also destabilize the DnaK-CMLA
complex (Palleros et al., 1993).
Figure 4:
Isolation of DnaK-CMLA and DnaK-P
complexes using size exclusion HPLC on Superdex 200 column. DnaK
protein (35 µg) was preincubated with CMLA (15 µg, panel
A) or
P (26 µg, panel B) in buffer B (50 µl)
for 30 min at 30 °C in the presence or absence of ATP, AMP-PCP, or
ADP (1 mM) and injected onto a Superdex 200 column
equilibrated with buffer B. In the case when DnaK was preincubated with
P and ATP, 200 µM ATP was also present in the column
buffer. Bottom HPLC profiles represent control experiments where DnaK
protein or
P protein alone were chromatographed under the same
conditions (column buffer supplemented with 200 µM ATP).
The Superdex 200 column was calibrated with the following Bio-Rad
molecular weight standards: 1, thyroglobulin (670 kDa); 2, bovine
-globulin (158 kDa); 3, chicken
ovalbumin (44 kDa); 4, equine myoglobin (17.5
kDa).
Figure 5: Interaction of the DnaJ with DnaK or DnaKc94 in the presence of nucleotides. The DnaJ or BSA (0.5 µg/well) was loaded on the ELISA plate wells in PBS buffer and following binding, washing, and blocking procedures, as described under ``Materials and Methods,'' the increasing amounts of DnaK or DnaKc94 protein in buffer A/BSA, in either the absence or presence of ATP (1 mM) and AMP-PCP (1 mM), was added. The reaction was preincubated for 30 min at room temperature and cross-linked with glutaraldehyde as described under ``Materials and Methods.'' The amount of DnaK protein bound to DnaJ was estimated using an anti-DnaK serum as described under ``Materials and Methods.''
To verify the results obtained with
ELISA we performed size exclusion HPLC. Under the described conditions
the DnaK protein eluted as an asymmetric monomer (Montgomery et
al., 1993). The DnaJ protein behaved as a dimer, as
described previously (Zylicz et al., 1985). When both proteins
were mixed together, in the absence of ATP, their elution profiles were
unchanged, i.e. there was no detectable interaction (Fig. 6). When ATP was added during the preincubation there was
no noticeable interaction (Fig. 6). However, when ATP was
present in both the premixture and in the column buffer, we did observe
an additional peak, suggesting that the DnaK and DnaJ proteins interact
in an ATP-dependent manner. To confirm this interpretation we separated
the fractions from the HPLC sizing column by SDS-polyacrylamide gel
electrophoresis and verified that only in the presence of ATP in both
the premixture and column buffer did the DnaK and DnaJ proteins
co-elute in these fractions (results not shown). The estimation of the
apparent molecular weight of this putative DnaJ-DnaK complex suggests
that it is slightly smaller than a dimer of DnaK. Thus, we conclude
that a DnaJ dimer (or monomer) interacts with a DnaK monomer (Fig. 6).
Figure 6:
Detection of a DnaK-DnaJ complex. The DnaK
(35 µg) was incubated with DnaJ (40 µg) in the absence or
presence of ATP in the premixture (DnaK, DnaJ, and ATP); in the
presence of ATP both in the premixture and the column buffer (DnaK,
DnaJ, and ATP/ATP) and in the presence of ATP in the premixture and ADP
in the column buffer (DnaK, DnaJ, and ATP/ADP). Control experiments
show the chromatography profile of either DnaK or DnaJ alone in the
presence of ATP in the premixture and column buffer (DnaK, ATP/ATP and
DnaJ, ATP/ATP, respectively). The concentration of ATP or ADP was 200
µM. The position of dimer DnaK (2K) was estimated
by chromatographing the DnaK protein (10 mg/ml). At that concentration,
the DnaK dimer form is easily detectable (Montgomery et al.,
1993). The Superdex 200 column equilibrated with buffer B was
calibrated with the following Bio-Rad molecular weight standards: 1, thyroglobulin (670 kDa); 2, bovine -globulin
(158 kDa); 3, chicken ovalbumin (44 kDa); 4, equine
myoglobin (17.5 kDa).
The substitution of ATP by ADP (or AMP-PCP) in the column buffer did not lead to the formation of a DnaK-DnaJ complex (result not shown), suggesting that the DnaK-ADP form derived from ATP hydrolysis is required for a stable DnaK-DnaJ interaction.
It was previously suggested that Hsp70 and DnaJ-like proteins
(Hsp40) exhibit different substrate specificity (for review, see Cyr et al.(1994)). It is generally assumed that the Hsp70 class of
protein binds to unfolded proteins, recognizing short polypeptides that
are in an extended configuration. By contrast, it is believed that
DnaJ-like proteins bind protein substrates in the ``molten
globule'' state exhibiting secondary and some tertiary structures.
While the binding of Hsp70 to different stretched polypeptides was
carefully examined (Flynn et al., 1991; Blond-Elguindi et
al., 1993a; Gragerov et al., 1994), no corresponding
systematic study of binding of Hsp70 to a native protein substrate nor
that of the DnaJ chaperone to native proteins or peptide substrates was
done. In this paper we used four, native substrates, O,
P,
, and RepA, as well as a permanently unfolded
polypeptide which lacks secondary structure and cannot fold, CMLA, to
compare the ability of these substrates to form a complex with the DnaK
or DnaJ molecular chaperones. We found that the DnaK protein,
previously assumed to preferentially recognize unfolded peptides in the
extended configuration, binds much better to native RepA then denatured
CMLA (or heat inactivated luciferase).
Perhaps these
seemingly conflicting observations with DnaK can be explained if one
assumes that the RepA native protein exhibits (transiently?) a
polypeptide loop(s) on the surface in the stretched configuration. On
the other hand, DnaJ chaperone, previously supposed to recognize
protein substrates with secondary and tertiary structure, indeed
recognizes efficiently the native
and RepA proteins
but binds with very low affinity to either the native
O,
P
proteins, or to the denatured CMLA. Perhaps
and
RepA exhibit such a protein structure on their surface that DnaJ likes
to bind to. What amino acid sequence or tertiary structure is
recognized by DnaJ protein remains still unknown.
In previously
published studies it was assumed that ADP, ATP, and ATPS all bind
to Hsp70 with comparable affinities, in the micromolar range (Palleros et al., 1991, 1993) This belief was recently challenged by
other laboratories (Prasad et al., 1994; Montgomery et
al., 1993). Taking into account the recently published data for
the affinity of DnaK to ATP (K
35
nM) and to ADP (K
200
nM) (Montgomery et al., 1993) most of DnaK (under
physiological concentrations of these two nucleotides) would be in the
nucleotide bound form. In the absence of the DnaJ protein, the DnaK in
the presence of ATP possesses the highest affinity toward a substrate
(high on rate), but exhibits an even higher off rate, as shown by using
the stopped-flow technique (Schmid et al., 1994). In this
paper we show that the presence of nonhydrolyzable ATP analogues can
``trap'' DnaK in a conformation that promotes efficient
DnaK-substrate complex formation. However, we cannot exclude the
possibility that for some protein substrates, binding to DnaK will also
accelerate the ATP hydrolysis step and/or nucleotide exchange. Such a
possibility was previously suggested for the eukaryotic Hsc70 by
Hightower et al.(1994). It is also possible that in some
cases, the binding of substrate to DnaK, which may induce
conformational changes in the chaperone (Park et al., 1993),
will also trigger the release of ATP or ADP from the DnaK chaperone.
Contrary to the previously published data (Palleros et al., 1991, 1994) we show in this paper that the presence of ADP in the premixture does not substantially accelerate the binding of DnaK to its protein substrate. Moreover, the hydrolyzable ATP leads to the dissociation of DnaK from substrate complex, as it was shown previously (Liberek et al., 1991b). We conclude that the DnaK-ADP form which is created after ATP hydrolysis possesses limited affinity to the protein substrates. As it will be discussed in the accompanying paper (Wawrzynów et al., 1995b), in order to stabilize DnaK-substrate complex, DnaK protein needs to be in the active conformation which can occur in the presence of DnaJ and ATP hydrolysis.
We previously showed that the presence of DnaJ accelerates (even without GrpE) the hydrolysis of ATP bound to DnaK (Liberek et al., 1991a; Szabo et al., 1994). In this paper we show that in the presence of hydrolyzable ATP it is possible to isolate DnaK-DnaJ complex. The presence of nonhydrolyzable ATP analogue, which promotes efficient binding of DnaK to its various protein substrates, does not stimulate DnaK-DnaJ complex formation. Our results suggest that DnaJ protein binds to the DnaK-ADP form only when this form is a product of DnaK-dependent ATP hydrolysis.
In summary,
in this paper we show that the DnaK-ADP form possesses low affinity to
the substrates but high affinity to the DnaJ protein. These results
suggest that the mechanisms of DnaK binding to DnaJ and DnaK binding to
substrate are fundamentally different. It is also possible that the
DnaK protein domains which are involved in the interaction with a
substrate and DnaJ are different. In preliminary experiments we have
shown that a DnaK mutant variant, lacking 94 amino acids from its
C-terminal domain (DnaKc94) does not interact stably with the DnaJ
protein, but does form a functional (but unstable) complex with the
P protein (this paper). (
)These results suggest that
DnaJ may interact stably with DnaK through DnaK's C-terminal
domain. Such a possibility was previously suggested by Tsai and
Wang(1994). However, more experiments are needed to verify this
hypothesis. For example, it is still possible that the truncation of
the C-terminal domain of the DnaK protein induces a conformational
change of the N-terminal domain of DnaK protein, thus preventing
formation of the DnaJ-DnaKc94 complex.