(Received for publication, May 22, 1995; and in revised form, September 20, 1995)
From the
DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is
an ATP-dependent chaperone whose basal ATPase is stimulated by
synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE.
We have used three mutant DnaK proteins, E171K, D201N, and A174T
(corresponding to Glu, Asp
, and
Ala
, respectively, in bovine heat stable cognate 70) to
probe the ATPase cycle. All of the mutant proteins exhibit some
alteration in basal ATP hydrolysis. However, they all exhibit more
severe defects in the regulated activities. D201N and E171K are
completely defective in all regulated activities of the protein and
also in making the conformational change exhibited by the wt protein
upon binding ATP. We suggest that the inability of D201N and E171K to
achieve the ATP activated conformation prevents both stimulation by all
effectors and the ATP-mediated release of GrpE. In contrast, the defect
of A174T is much more specific. It exhibits normal binding and release
of GrpE and normal stimulation of ATPase activity by DnaJ. However, it
is defective in the synergistic activation of its ATPase by DnaJ and
GrpE. We suggest that this mutant protein is specifically defective in
a DnaJ/GrpE mediated conformational change in DnaK necessary for the
synergistic action of DnaJ + GrpE.
The 70-kDa heat shock proteins (hsp70s) ()comprise a
ubiquitous family of essential proteins whose synthesis is induced by
increases in temperature and other forms of
stress(1, 2, 3, 4, 5) . A
vast collection of literature now supports the notion that a major
function of the hsp70 proteins is to interact with substrate proteins
to alter or maintain their
conformation(6, 7, 8, 9) . Binding
and hydrolysis of ATP by hsp70s is central to their function as
chaperones. The intrinsic ATPase of the hsp70s is modulated by binding
substrate proteins and, at least in bacteria, by interacting with their
cohort chaperones, DnaJ and GrpE (10) .
DnaK is one bacterial homolog of this conserved protein class. It shares 50% amino acid identity with its hsp70 family members (11) and exhibits a number of similar biochemical properties, including a high binding affinity for ATP, a weak ATPase activity(12) , and chaperone function(13, 14, 15) . The chaperone activity of DnaK presumably mediates its participation in a diverse spectrum of cellular processes including host, phage, and plasmid DNA replication, cell division, proteolysis, flagellar biosynthesis, translocation of secretory proteins, and regulation of the heat shock response(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) .
In order to understand the mechanism of action of DnaK, we isolated
two classes of partially defective dnaK mutants: recessive
mutants selectively defective in regulation of the heat shock response,
and dominant negative mutants(30) . Because these mutants
retain partial function, they are unlikely to result from gross
structural alterations and are suitable for a structure-function
analysis of DnaK. Three of the mutant proteins were of particular
interest because they were defective in a variety of cellular processes
even though they retained the ability to bind ATP. The two dominant
mutations, E171K (Glu in hsc70) and D201N (Asp
in hsc70), affect residues in the ATPase domain predicted to be
in the vicinity of the Mg
ion bound to ATP. Both were
suggested to be candidate residues participating directly in
catalysis(31, 32) . The recessive mutation, A174T
(Ala
in hsc70), lies in proximity (in the linear
sequence) to amino acids expected to participate directly in ATP
hydrolysis; no inferences were made concerning a role of Ala
in catalysis.
The availability of these three DnaK mutant
proteins that are able to bind ATP but are altered at amino acids
suggested to be critical for ATP hydrolysis led us to perform a
detailed characterization of their intrinsic and regulated ATPase
activities. Interestingly, the same residues identified by our dominant
negative mutations were chosen by two different groups for
site-directed mutagenesis. The work reported here complements recent
studies on the behavior of a series of mutant proteins altered at
Glu in DnaK(33) , as well as Glu
and Asp
in the N-terminal domain of
hsc70(34) . On the basis of our analysis, we suggest a pathway
for the regulated ATPase activity of DnaK.
Plasmid pNRK416 expressing wild
type dnaK from the
isopropyl--D-thiogalactopyranoside-inducible lacUV5
promoter was from N. Kusukawa and T. Yura, while synthetic vsv-peptides
A and C (>95% pure) (35) were synthesized by the
Biomolecular Resource Center at University of California, San
Francisco.
Cell pellets
(typically 5 g) were resuspended in 115 ml of buffer A supplemented
with 0.3 mg/ml lysozyme. Cell suspensions were lysed by repeated quick
freezing and thawing cycles followed by sonication. Cell debris and
unlysed cells were removed by centrifugation at 30,000 g for 45 min.
Peak fractions containing DnaK were pooled, dialyzed against buffer C and chromatographed through an ATP-agarose column (Sigma). The sample was allowed to interact with the column matrix and then washed with buffer C containing 2 M NaCl to eliminate proteins bound nonspecifically to the resin. The column was reequilibrated with buffer C and washed with 3 column volumes of buffer supplemented with 1 mM GTP to elute GTPases that may have bound to the column. To avoid the possibility of other lower affinity ATPases contaminating the preparation of DnaK, the column was next washed with buffer C containing 0.2 mM ATP. DnaK bound to the column was finally eluted with buffer C containing 10 mM ATP. Although DnaK was expected to elute predominantly in buffer containing 10 mM ATP, at least 50% of it was present in the 0.2 mM ATP eluate. A likely explanation for this behavior is that the 2 M salt wash may have weakened the interaction between DnaK and ATP-agarose allowing it to be eluted with lower ATP concentrations. Peak fractions of DnaK in the 0.2 mM and 10 mM ATP eluates were pooled separately. Each pool was concentrated using a Centricon 30 concentrator and free ATP was removed from the protein solution by extensive dialysis against buffer D.
The 0.2 mM eluate was further purified on a hydroxylapatite (Bio-Rad) column. This additional step also ensured removal of free ATP, left over after dialysis following ATP-Sepharose chromatography. Samples were diluted with buffer E to adjust the conductivity of the DnaK solution. DnaK was retained on the column under these conditions and was eluted with a linear gradient of sodium phosphate ranging from 10 mM to 120 mM in buffer E. Peak fractions containing DnaK were concentrated and dialyzed against buffer D containing 50 mM KCl. The dialyzed fractions were aliquotted, quick frozen in dry ice-ethanol, and stored at -80 °C. Protein concentration was measured by the dye binding method using Coomassie Brilliant Blue G-250 (Bio-Rad protein assay kit), with bovine serum albumin as a standard. All preparations were greater than 95% homogeneous after elution from the ATP-Sepharose column.
0.8 mg His-GrpE (in buffer H) was
coupled to 0.2 ml (settled volume) of Ni-NTA resin
(QIAGEN) by incubation at room temperature for 2 h. Coupling was
monitored by doing a Bradford measurement of the starting solution and
comparing it with that of the supernatant after incubation (>90% of
the protein was bound to the resin under these conditions). The resin
was equilibrated in buffer H and 10 µg of each of the DnaK
proteins, dialyzed against the same buffer, were batch adsorbed with 10
µl (settled volume) of either Ni
-NTA or
His-GrpE-Ni
-NTA resin (corresponding to
40
µg of bound GrpE). After a 1-2 h incubation at room
temperature, the resin was centrifuged and the supernatant,
corresponding to the flow-through fraction, was saved in a separate
tube. The resin was washed twice in buffer D and then incubated in the
presence of either 0.2 mM or 1 mM ATP in buffer D at
30 °C for 30 min. The resin was respun, and the supernatant (or the
eluate) was transferred to a new vial. Aliquots of the onput,
flow-through, and eluate fractions were electrophoresed on 10%
SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue to
detect the protein pattern.
We purified wild type and three mutant DnaK proteins, E171K,
A174T and D201N, to apparent homogeneity by a three-step process
involving chromatography over DEAE-cellulose, ATP-Sepharose, and
hydroxylapatite column matrices. Because these mutations were expected
to affect ATP hydrolysis by DnaK, we compared their basal and regulated
ATPase activities with those of wt DnaK. Initially, two different
techniques were used to quantitate the ATPase activity. The amount of
either the free phosphate (P) released or ADP formed was
measured by scanning polyethyleneimine cellulose sheets following thin
layer chromatography. Alternatively, the free phosphate was complexed
with molybdenum and extracted using H
O-saturated 1-butanol
(see ``Experimental Procedures''). All measurements gave
identical results (data not shown). However, due to the sensitivity and
rapidity of manipulations, the molybdenum assay was used in all
experiments reported in this study.
As we were finishing our studies, we became aware
that our data with E171K differed significantly from those of the Bukau
group(33) . Whereas our preparation of the E171K mutant protein
exhibited lower ATPase activity than the wt protein (Table 1),
theirs exhibited higher than normal activity. Differing purification
procedures or assay conditions could have accounted for this
discrepancy. Systematic analysis of these variables indicated that the
assay conditions were responsible. The ATPase of E171K was 44-fold
higher in buffer G used by Bukau than in our buffer, buffer F (Table 2). This dramatic effect was specific for the E171K
mutant. The activity of wt DnaK was identical in both buffer systems
and those of A174T and D201N increased only 2-fold in buffer G (Table 2).
To identify the component that contributed to the
increased activity of E171K, we switched individual components of both
buffer systems. These experiments indicated that acetate was the key
difference in the two buffers (Table 3). Replacing Mg(OAc) in buffer G with MgCl
reduced the activity of E171K
to near background. Supplementing buffer G containing MgCl
with KOAc restored the ATPase of E171K activity to a level
comparable to that observed in buffer G only, indicating that
Cl
was not exerting a deleterious effect. Conversely,
replacing MgCl
in buffer F with Mg(OAc)
raised
the activity of E171K almost to the level observed in buffer G. The
effect was specific to acetate since glutamate did not affect the
activity of E171K (data not shown). These results indicate that acetate
specifically alleviates the adverse effect of the lysine substitution
in the E171K mutant protein. All the experiments reported in this study
were performed in buffer G, unless otherwise indicated.
Figure 1:
Mutant DnaK proteins
have increased K values for ATP. DnaK
proteins (0.6 µM, except for A174T which was at a
concentration of 1.2 µM) were incubated in buffer G at 30
°C for 60 min (Panel A, wt DnaK), 40 min (Panels B and D, D201N and E171K, respectively) or 80 min (Panel C, A174T). The ATP concentrations ranged from 0.3 to 25
µM (wt DnaK), 0.5 µM-50 µM (D201N), 0.5 µM to 0.4 mM (A174T) and 0.5
µM to 1.0 mM (E171K). Independent experiments
established that the rate of hydrolysis was linear with time for each
condition. Picomoles of ATP hydrolyzed per min (V) were
calculated for each protein at every ATP concentration; the double
reciprocal, 1/V versus 1/ATP, plots were used to derive K
values.
Figure 3:
Interaction of wt and mutant DnaK proteins
with GrpE. 10 µg of each DnaK protein was mixed with either
Ni-NTA resin (Panel A) or with
His-GrpE-Ni
-NTA resin (Panel B). Following
incubation at room temperature for 1-2 h, the resins were
centrifuged, and the supernatants (or flow-through fractions, FT) were saved separately. The resins were then incubated with
0.2 mM ATP (for wt DnaK, A174T, and D201N) or 1 mM ATP (for E171K) at 30 °C for 30 min. The resins were
recentrifuged, and the ATP eluates were likewise transferred to a
separate tube. Aliquots of the starting material, flow-through
fractions, and ATP eluates were electrophoresed through 10%
SDS-polyacrylamide gels; the profile of DnaK in these fractions was
visualized by Coomassie Blue staining. The lower band reflects GrpE,
which appears to show a slow elution from the column under these
conditions.
Figure 2:
Effect of synthetic peptides on the ATPase
activity of wt and mutant DnaK proteins. Purified DnaK proteins were
incubated in the absence (-
) or presence of 0.5
mM peptide A (
-
) or peptide C
(
-
) at 30 °C with saturating
concentrations of [
-
P]ATP. For wt DnaK,
A174T, and D201N, this corresponded to 0.2 mM ATP; for E171K,
the ATP concentration used was 0.75 mM. At indicated times,
25-µl reaction aliquots were assayed for ATP hydrolysis as
described. A, wt DnaK; B, A174T; C, E171K;
and D, D201N.
A174T responded similarly to wt DnaK to the separate addition of either DnaJ or GrpE. However, DnaJ and GrpE did not function synergistically to increase the ATPase activity of A174T. Together, DnaJ and GrpE stimulated the A174T ATPase with less than twice the effectiveness of DnaJ alone. In contrast, for wt DnaK, DnaJ and GrpE together were approximately 25-fold more effective in stimulating the ATPase activity than DnaJ alone.
The ATPase activity
of E171K was not stimulated either by the separate or simultaneous
addition of DnaJ or GrpE. In addition, there was no stimulation by DnaJ
± GrpE when the experiments were carried out in buffer
containing MgCl rather than Mg(OAc)
where the
intrinsic ATPase activity of E171K was reduced by more than 40-fold
(data not shown). As was true with E171K, neither DnaJ, alone, nor DnaJ
+ GrpE stimulated the ATPase activity of D201N. Moreover, GrpE
reduced the ATPase of D201N by as much as 40% (Table 4). Though
small, the inhibitory effect of GrpE was reproducible.
wt DnaK and all three mutant proteins bound specifically to the
his-GrpE column. Whereas >90% of the DnaK was present in the flow
through fraction when chromatographed over the Ni-NTA
resin alone (Fig. 3, Panel A), only trace amounts were
detectable in the flow through fractions of the
His-GrpE-Ni
-NTA resin (Fig. 3, Panel
B). Thus, the mutants were not grossly defective in their
interaction with GrpE. We note in passing that the ATPase activity in
the flow-through of the His-GrpE-Ni
-NTA resin was
proportional to the small amount of DnaK present in that fraction,
indicating that the activities we have measured throughout this report
are attributable to DnaK, rather than to a contaminating ATPase.
In
contrast to the binding phase, the ATP elution phase revealed
differences between wt and mutant proteins (Fig. 3, Panel
B). As expected, wt DnaK was efficiently eluted by 0.2 mM ATP; A174T behaved similarly to the wt protein. In contrast, only
10-20% of E171K and D201N were recovered. ATP concentrations as
high as 1 mM were unable to further elute E171K from the bound
resin. ()These data suggest that both E171K and D201N are
defective in the ATP mediated conformational change in DnaK necessary
for release of GrpE.
Figure 4: Stability of wt and mutant DnaK proteins in buffer I at 30 °C. 3 µg of DnaK were incubated in buffer I at 30 °C in the absence or presence of 5 mM ATP. The incubations were terminated at the indicated time periods by the addition of Laemmli sample loading buffer. Partial proteolytic products were resolved by electrophoresis on 12% SDS-polyacrylamide gels and visualized by staining with Coomassie Blue R-250 .
The differences in digestion of wt DnaK with and without nucleotide have been carefully documented (48, 49) and were reproduced here (Fig. 5A). In the absence of ATP, wt DnaK exhibited five predominant proteolytic products of about 55, 46, 44, 31, and 17 kDa in size. In the presence of ATP, proteolysis was considerably accelerated and the predominant digestion products were altered. The 55-, 44-, and 17-kDa fragments remained, the 31-kDa fragment disappeared, and fragments of 53 and 45 kDa became prominent. The 45-kDa fragment, which is the major band generated upon prolonged digestion in the presence of ATP, corresponds to the N-terminal ATPase domain of DnaK and may be distinct from the 46-kDa band observed in the absence of ATP(33, 34) .
Figure 5: Partial proteolysis of wt and mutant DnaK proteins. 3 µg DnaK were incubated with TPCK-trypsin in buffer I at 30 °C in the absence or presence of 5 mM ATP. The digestions were terminated and analyzed as described in the legend to Fig. 4. wt DnaK (Panel A), A174T (Panel B), D201N (Panel C), and E171K (Panel D).
The pattern of proteolytic products generated by A174T was almost identical to that of wild type DnaK both in the absence and presence of ATP (Fig. 5B). Thus, at the level of sensitivity of this assay, both the native structure of A174T and its conformational change upon binding ATP are virtually identical to that of the wt protein.
For D201N, the tryptic peptides in the absence of ATP were almost identical to wt (Fig. 5C). However, addition of ATP induced very little change in either the rate of appearance or pattern of tryptic peptides. These results suggest that although the conformation of D201N in the absence of ATP is similar to that of the wt, it does not exhibit the conformational change characteristic of wt DnaK upon nucleotide binding.
The partial proteolysis products of E171K differed most
from those of wt DnaK (Fig. 5D). E171K was much more
labile in the absence of ATP and generated peptides that were distinct
from those of wt DnaK even after limited extents of proteolysis. The
presence of ATP reduced the rate of trypsin digestion and in
particular, stabilized the 46-kDa ATPase fragment from degradation.
Since the ATPase activity of E171K was dramatically increased in the
presence of acetate ions relative to that in its absence, we wondered
whether the protein under went major structural changes in response to
acetate. We tested this by carrying out partial proteolysis of E171K in
buffer I containing MgCl instead of Mg(OAc)
.
Interestingly, the pattern of tryptic peptides generated without
acetate was virtually identical to that with acetate suggesting that
acetate may cause only local and/or subtle changes in the structure, if
any (data not shown). These results suggest that the conformation of
E17lK is discrepant from that of the wild type protein both in the
absence and presence of ATP.
The ATPase activity of DnaK is central to its mode of action
as a chaperone. The intrinsic ATPase activity of DnaK is very low (k
0.1-1.0 min
),
and physiologically important modulators, including denatured proteins
and the cohort heat shock proteins DnaJ and GrpE increase this basal
rate of hydrolysis. Both direct physical measurements and inferences
from homologous crystal structures support the idea that hsp70s undergo
conformational changes upon binding ATP. This conformational change is
crucial to chaperone activity as it alters the kinetics with which
hsp70 binds and releases substrate proteins. The availability of three
mutant DnaK proteins that retain the ability to bind ATP but are
defective in chaperone activity in vivo allowed us to explore
the relationship between the ATPase cycle and chaperone function. The
effects of each of these mutations on the intrinsic and modulated
ATPase activities are summarized in Table 4. Below, we discuss
how analysis of the effects of these mutational changes has altered our
view of the function of hsp70.
Glu (Glu
in bovine
hsc70) occupies a position in close proximity to the active site,
forming a hydrogen bond with a water molecule in the first coordination
shell of Mg
. Proper positioning of the Mg
ion, which interacts with the
-phosphate of ATP, is crucial
for catalysis. The Glu
position has been the subject of a
great deal of study because it has been hypothesized both to be a
catalytic base in the ATPase reaction and essential for the subdomain
movement that may couple ATPase activity with substrate binding. To
pursue these ideas, the McKay and Bukau groups made site-directed
changes at Glu
and examined various aspects of structure
and function of the mutant proteins. Our studies add additional
information concerning the function of this residue in both basal and
regulated ATPase activities of DnaK.
Glu is in a
structurally equivalent position to an aspartic acid proposed to be a
catalytic base in hexokinase, leading the McKay group to suggest
Glu
as one of four possible acidic residues that could
participate in catalysis (31, 32, 34, 49) . The ATPase
activities of N-terminal fragments of hsc70 containing either E175S or
E175Q substitutions are defective; the k
values
are 5-20-fold lower, and the K
values are
about two orders of magnitude greater than the
wt(34, 49) . However, both mutants retain ATPase
activity indicating that Glu
is not essential in
catalysis. Similar studies with E171A, E171L, and E171K substitutions
in DnaK also demonstrated that the mutant proteins exhibit significant
catalytic activity(33) . In fact, in contrast to the results
with the hsc70 mutants, the DnaK mutants exhibit V
values 3-30-fold greater than wt. Our results with E171K,
isolated using a different purification protocol, are in agreement with
the results of the Bukau group. ATP hydrolysis by E171K is about
13-fold higher than the wt rate. However, we find that the high k
is not an intrinsic property of E171K, but is
dependent on having acetate in the buffer. The presence of acetate
specifically enhances the rate of hydrolysis of E171K about 40-fold.
Glutamate cannot substitute for acetate indicating that a specific
interaction between E171K and acetate is involved in this effect. We
currently do not know whether the acetate effect is specific for the
E171K mutation or if it affects the k
of other
substitutions at residue Glu
. Moreover, we do not know
whether acetate also enhances the k
of hsc70.
The McKay assays were performed in acetate; however, if slight
structural differences prevented acetate enhancement of hsc70
catalysis, the k
differences of mutant hsc70 and
DnaK proteins would be explained. We consider three possible roles for
acetate in enhancing ATP hydrolysis by E171K: 1) acetate may perform
the function of the original glutamic acid residue, 2) binding of
acetate may alter the position of the lysine residue permitting greater
function, or 3) binding of acetate may specifically neutralize the
deleterious charge effects of the lysine residue. Examination of the
homologous crystal structure indicates that some possible conformations
of lysine could accommodate an acetate ion. (
)
Glu is located in one of two crossed
-helices connecting the two
N-terminal subdomains and is the only residue in these helices that
interacts with Mg
. In analogy to actin, Bukau and
collaborators (33) suggest that the two subdomains move upon
binding ATP using the crossed helices as a hinge and that this movement
requires a Mg
connection, thus implicating
Glu
in this process. Further correlates are that this
movement is essential for coupling ATPase activity to substrate binding
which, in turn, is essential for chaperone function. Consistent with
this idea, they find that E171A, E171L, and E171K mutant DnaK proteins
1) do not undergo the wt conformational change upon binding ATP as
judged by partial proteolysis, 2) bind peptides normally but exhibit
neither peptide-stimulated ATPase nor ATP-stimulated release of
peptides, and 3) are defective in chaperone activity. Our studies agree
with these findings and indicate that E171K is also defective in its
responses toward DnaJ and GrpE. Its ATPase can neither be stimulated by
DnaJ, nor synergistically increased by DnaJ + GrpE. Additionally,
the mutant protein is defective in ATP-mediated release from GrpE. Our
view of the origin of the E171K phenotype is somewhat more general than
that of Bukau. We believe that E171K is one example of a class of
mutations that interfere with the normal conformational change of DnaK
upon binding ATP and that the inability to make this conformational
changes interferes with all of the regulated activities of the protein.
This view is based primarily upon the phenotype of D201N and is
explicated below.
Like Glu,
Asp
(Asp
in bovine hsc70) occupies a
position in close proximity to the active site. Asp
is
coordinated via one carbonyl oxygen to the K
ion that
lies at the interface between protein and P
in the ADP form
of bovine hsc70(50) . In addition, Asp
is
coordinated via a water molecule to Glu
(50) .
Finally, like Glu
, Asp
by analogy to the
actin structure was suggested to act as a proton acceptor in catalysis
of ATP(34) . However, studies by the McKay group indicated that
altering Asp
to either Asn
or Ser
did not eliminate catalytic activity of the N-terminal fragment
of hsc70. In fact, of the four positions substituted by the McKay
laboratory, mutations at Asp
are the least defective; the
mutant proteins exhibit almost no alteration in K
and less than a 10-fold decrease in k
(34) . Our studies on D201N, performed on full-length DnaK,
support those of the McKay group in that we find only a small increase
in K
. However, reminiscent of the results with
Glu
, D201N also exhibits an increase in k
. In addition, crystallographic studies of
D206N and D206S demonstrate that the structure of the mutant proteins
bound to ADP is virtually identical to wt(49) . In summary,
D201N has only a very small effect on basal ATPase activity or on the
structure of the N-terminal fragment bound to ADP.
In stark contrast, D201N is severely defective in all of its regulated activities. Peptides bind normally to D201N, but do not stimulate its ATPase activity. On the contrary, they slightly, but consistently, inhibit the ATPase activity. Similarly, neither DnaJ nor DnaJ in combination with GrpE stimulate D201N ATPase. In fact, like peptide, GrpE also slightly inhibits the ATPase activity of D201N. Finally, as observed with E171K, D201N is defective in ATP-mediated release from GrpE.
Why does the D201N change completely abolish effector stimulation of the ATPase activity? The partial proteolysis phenotype of D201N may provide us with a possible explanation. Prior to binding ATP, the conformations of D201N and wt DnaK are indistinguishable as judged by partial proteolysis profiles. Following ATP binding, the wt protein exhibits significant qualitative and quantitative changes in partial proteolysis pattern, whereas D201N does not. This suggests that D201N is very defective in undergoing a conformational change after binding ATP. If the conformationally altered ATP bound form of DnaK (DnaK*) is the substrate with which DnaJ, GrpE, and peptide all interact productively, then D201N will be defective in all regulated activities because it cannot form DnaK*. Two studies, using completely different methodologies, indicate that ATP binding, without hydrolysis, alters DnaK so that it both binds and releases peptide more rapidly(9, 51) . These studies provide independent evidence for the idea that proper interaction with peptides requires DnaK*. Our explanation for the mutant phenotype suggests that productive interaction with DnaJ and GrpE requires DnaK* as well.
Our analysis indicates that
the mutation has a minimal effect on most of the activities we have
examined. The basal ATPase activity of A174T is basically intact. The
mutant protein exhibits a k about 50% that of
the wt and a heterogeneous K
for ATP. The lower K
of 1 µM is identical to that of the
wt, whereas the higher value is about 20-fold greater. However, given
the high intracellular ATP concentration (2.7
mM)(52, 53) , this heterogeneity is unlikely
to affect basal ATPase function. Likewise, the K
for peptide binding is unaltered relative to the normal value and
the ATPase of A174T is increased by two different substrate proteins.
Finally, as monitored by partial proteolysis, A174T and wt DnaK undergo
similar conformational changes in the presence of ATP.
The
outstanding characteristic of A174T is that the simultaneous addition
of DnaJ and GrpE does not result in a synergistic increase in its
ATPase activity. This is particularly surprising since assays for the
effects of the individual proteins on the A174T ATPase did not reveal
any defects. DnaJ stimulation of the A174T ATPase is equivalent to that
of wt. Likewise, binding and release of A174T to/from GrpE are normal.
The mutant phenotype could be explained if binding of DnaJ, GrpE, or
both proteins normally induces a conformational change in DnaK (to give
DnaK) that is required for the observed synergistic action of DnaJ
+ GrpE. The A174T mutation may prevent this conformational change
and abolish synergy. Ala is located in the same
-helix as Glu
(described above), one of the two
crossed
-helices in the hinge region connecting the N-terminal
subdomains. This region of DnaK is believed to be crucial in
conformational changes in response to effectors. Whereas Glu
is at the N terminus of this helix and points into the active
site, Ala
lies in the middle of the helix facing a large
-sheet in subdomain 2A(33) . To examine the structural
consequences of altering this alanine to threonine, we modeled the
A174T change into the known structure of hsc70, where the corresponding
residue is Ala
(Fig. 6). Substitution of threonine
for alanine results in a small but significant steric overlap between
the threonine methyl group and valines 335 and 337 of the adjacent
-sheet, which implies either distortion or displacement of the
Ala
-helix relative to the
-sheet. In addition,
this threonine is now in close proximity to Val
in the
other crossed
-helix connecting the two subdomains. The extra bulk
in the vicinity of the hinge region is likely to interfere with a
number of possible conformational rearrangements involving the two
crossed helices and the associated
-sheet.
Figure 6:
Model of Ala
Thr in
the context of the hsc70 structure(31) . The residues in this
figure are numbered according to hsc70: hsc70 Ala
corresponds to Ala
, hsc70 Glu
corresponds
to Glu
, and hsc70 Asp
corresponds to
Asp
. Thr
(T179) indicates the
position of the Ala
Thr substitution. ADP,
phosphate, and Mg
are shown in blue, protein
backbone is shown in yellow. Selected side chains shown in CPK
colors (O, red; N, blue; C, gray). A, overview, showing the
crossed helices below the ATP binding cleft: one helix includes
Thr
(T179) (Thr
in DnaK), the
other crosses and continues down to the lower right. B,
close-up of the environment of Thr
. Substitution of
threonine for alanine leads to steric clashes with valines 335 and 337
when the methyl hydrogens are taken into account. Note that Ile
(I197) appears closer to Thr
in this
figure, but is actually behind the plane of the threonine. The
corresponding alanine residue is found in all 22 known bacterial DnaKs
and 74 of 78 eukaryotic hsc70 homologs.
It has recently been
suggested that DnaJ induces a conformational change in DnaK. Studies of
partial DnaJ proteins indicate that the ability to stimulate ATPase and
cause the conformational change reside in partially distinct regions of
the DnaJ protein(54) . This conformational change is required
for the tight binding of DnaK to which is a
prerequisite for proper regulation of the heat shock
response(54) . If A174T were unable to make this conformational
change, the defect of A174T in regulating the heat shock response could
be explained. It is currently unknown whether a similar tight binding
state is required for efficient participation of DnaK in
replication.
Our evidence that the DnaK* form of the protein is required for all subsequent regulated changes in the ATPase cycle comes from the study of two mutant proteins, E171K and D201N. This evidence is particularly compelling for D201N, which exhibits a normal conformation in the absence of ATP but fails to achieve the conformationally altered nucleotide bound form of DnaK. In addition, D201N fails to perform all regulated activities of the DnaK protein that we have tested. The simplest way to explain this mutant phenotype is to suggest that the conformationally altered DnaK* form of the protein is required for all regulated activities including: 1) stimulation of the ATPase activity of DnaK by peptides, DnaJ, or DnaJ + GrpE and 2) ATP-mediated release of GrpE from DnaK. DnaK* could be required either for effector binding or for signal transmission. For peptides and GrpE, the DnaK* form must be required for signal transduction, since both ligands can bind to DnaK. For DnaJ, it is not known whether DnaK* is required for binding or signal transduction.
Our evidence that the DnaK form of the protein is required for synergistic activation of DnaK by DnaJ and GrpE comes from the study of the third mutant protein A174T. A174T binds both DnaJ and GrpE normally but is specifically defective in the synergistic stimulation of ATPase caused by the simultaneous binding of these effector molecules. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK (to give DnaK), and that this conformational change is necessary for the synergistic action of DnaJ + GrpE. The biochemical phenotypes of intragenic second site revertants of each mutation may test the validity of these ideas.