(Received for publication, June 20, 1995; and in revised form, September 7, 1995)
From the
In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.
The heat shock protein 70 (HSP70) ()family of
chaperones are components of the cellular machinery for folding,
assembly, and degradation of proteins(1) . These proteins are
thought to undergo cycles of nucleotide-mediated binding and release to
unfolded polypeptides. The binding of peptides to HSP70 proteins
stimulates their ATPase activity(2) , and bound peptides or
proteins are released with ATP but not with non-hydrolyzable
analogues(2, 3, 4, 5) . These
observations have led to the conclusion that ATP hydrolysis is required
for HSP70 activity and that there are functional interactions between
the ATP binding and protein binding domains. All HSP70 proteins bind
ATP tightly but the ATP hydrolysis rates of purified proteins are so
low under physiological conditions, that other co-factors may be
required to enhance the rate of ATP hydrolysis. In bacteria two
co-factors, dnaJ and grpE, have been identified that together increase
the ATPase activity of dnaK (bacterial HSP70 homologue) up to 50-fold (6) . Although dnaJ homologues have been identified in several
organelles in various organisms(7) , the only eukaryotic grpE
homologues found thus far are mitrochondrial(8, 9) .
Alternatively, it is possible that ATP binding rather than ATP
hydrolysis is essential for HSP70 function. In support of this
hypothesis, investigators have recently demonstrated that peptides are
released from both dnaK and hsc70 (mammalian cytosolic homologue) after
ATP binding but before ATP hydrolysis occurs(10) . Thus, it is
presently unclear whether ATP binding, ATP hydrolysis, or both are
important for HSP70 function in vivo.
The ATP binding
domain of all HSP70 members resides within a highly conserved
NH-terminal 44-kDa fragment that can be generated by
proteolysis(11, 12) . The structure of the ATP binding
domain of hsc70 has been solved in the presence of ADP (13) and
AMPPNP (14) , allowing investigators to identify residues that
might participate in either ATP binding or hydrolysis. To date, a
number of mutations have been made in the active site of the various
HSP70 family members(15, 16, 17) , and these
have been characterized to varying degrees.
A series of dnaK
(bacterial HSP70) and Kar2 (yeast endoplasmic reticulum HSP70) mutants
have been isolated by genetic screens that have mutations mapping to
their ATP binding domain(17, 18) , but most of them
have not been characterized biochemically. Mutation of the
autophosphorylation site (Thr) of dnaK, yielded mutants
that had greatly reduced ATP hydrolysis rates (19) and that
were unable to complement dnaK null bacterial strains(20) .
Several mutations have been made in the active site of the ATPase
domain of bovine hsc70(15, 21) . Biochemical
characterization of Thr
mutants (corresponding to
Thr
in dnaK) revealed that the mutation increased the K
for ATP(21) , while mutation of
four other active site residues (Asp
, Asp
,
Glu
, and Asp
) decreased the k
values. Mutation of two of these (Asp
and Glu
) resulted in increased K
values for ATP(15) . Interestingly, Glu
corresponds to one of the residues identified in the dnaK genetic
study(17) . No functional data are available on the effects of
the various hsc70 mutants. Several point mutations in the ATP binding
domain of hamster BiP have been made(16, 22) ,
including Thr
(corresponding to Thr
in dnaK
and Thr
in hsc70) and Glu
(corresponding to
Glu
in dnaK and Glu
in hsc70). These
mutations affected the ATPase activity of BiP but did not inhibit their
ability to bind to ATP-Sepharose or to bind to substrate
proteins(16) . Transient expression of the BiP ATPase mutants
in mammalian cells causes a disruption of the endoplasmic reticulum (23) and interferes with the folding of a nascent endoplasmic
reticulum protein. (
)A rigorous biochemical characterization
of these mutants has not been done.
Thus, there is a growing amount of data to demonstrate that either the ATP binding or hydrolysis properties of HSP70 proteins are important in their in vivo function, but in most cases, ATP binding site mutants have not been characterized in enough detail to determine the role of nucleotide in regulating HSP70 activity. In this study, we have produced a series of single point mutants in the ATP binding site of hamster BiP and purified these recombinant proteins from bacteria. Mutants that retained the structural integrity of wild-type BiP were selected for further characterization in terms of ATPase activity, nucleotide binding affinity, and the ability of nucleotide to induce a conformational change in the mutant protein. Through these characterizations, we were able to classify the ATP binding site mutants into three separate groups: ATP binding mutants, ATP hydrolysis mutants, and conformational change mutants. The mutants were used to determine the requirements for the in vitro release of bound peptides.
Figure 1:
Mutated residues in BiP were located in
the ATP binding site of hsc70 to demonstrate their spacial relation to
ATP. Presumed positions of the mutated residues in BiP are depicted
based on the known position of the homologous residues in hsc70. For
each residue, the actual position of the hsc70 side chain is shown with
the BiP residue number (i.e. T37 = Thr in
hsc70, G226 and G227 = Gly
and
Gly
in hsc70, and T229 = Thr
in hsc70). Structural manipulation was performed by using Rasmol
program.
Two dominant negative mutations have been identified in Kar2 (yeast
BiP homologue) that occur at residues corresponding to Gly and Gly
in hamster BiP. (
)In both cases,
the mutation involved a glycine to aspartic acid change. The
corresponding residues were identified on the three-dimensional
structure of the hsc70 ATPase domain (Fig. 1), and the distances
between the
carbon of these glycines and the oxygen group of the
phosphate of ADP were measured. Gly
was 3.66
Å from ADP and Gly
was 4.09 Å away,
suggesting that the substitution of an amino acid with a large charged
side chain at either position might have profound effects on nucleotide
binding or hydrolysis. All of these structurally and genetically
identified residues (Thr
, Thr
,
Glu
, Gly
, and Gly
in BiP) are
completely conserved in Escherichia coli, yeast, and mammalian
HSP70 family members.
Figure 2:
Nondenaturing gel analysis of mutant BiP.
15 µg of Ni-agarose purified, recombinant hamster
BiP proteins were electrophoresed on 2-15% nondenaturing
polyacrylamide gel at 4 °C for 16 h. The proteins were then
visualized by Coomassie Blue stain. The positions of monomers, dimers,
and higher order oligomers are marked on the left.
Figure 3:
ATPase activity of BiP mutants. The ATPase
activity of wild-type and mutant BiP proteins were measured using 2.0
µM protein and 1.0 mM [-
P]ATP in the standard ATPase/ATP
binding buffer (``Materials and Methods''). The reactions
were incubated at 37 °C for the indicated time, and 20 µl were
removed for
P extraction and counted. Nanomoles of P
liberated were calculated based on the specific activity of the
[
-
P]ATP. Bovine serum albumin was assayed
in parallel as a negative control. The range of values obtained from
various preparations of each mutant is provided for the 30-min time
point.
Similar studies on the G226D and G227D mutants revealed that,
although both mutants retained some ATPase activity (K
100 µM), we were unable to measure nucleotide
binding by equilibrium dialysis using nucleotide concentrations ranging
from 50 to 400 µM with 50 µM of mutant
protein. These binding data were further supported by HPLC analyses of
the nucleotide content of these mutants after they were purified from
bacteria. Unlike WT BiP and the T229G mutant, which both contained
bound ADP that was resistant to dialysis, the G226D and G227D mutants
had no detectable nucleotide bound to them (data not shown).
Thus,
we were concerned that the ATPase activity observed with G226D and
G227D could be due to a trace contamination by a stronger ATP
hydrolyzing protein. To rule out the possibility of contamination, we
first rebound the recombinant protein to Ni-agarose
and washed the column with 1 M salt to remove any
BiP-associated proteins, and second, we chose several new bacteria
colonies expressing each of the mutants. Both the salt-extracted and
the new isolates were analyzed for ATPase activity and nucleotide
binding. Identical results were obtained: the G226D and G227D mutants
still had some ATPase activity but no detectable nucleotide binding
capability. Photoaffinity labeling of the G227D mutant with 100
µM radioactive azido-ATP demonstrated that this mutant
could indeed bind ATP (data not shown), further ruling out the
possibility that the ATPase activity came from a contaminating protein.
Thus, these two mutants evidently bind ATP, but their affinity for ATP
is decreased to such an extent that the binding assay used here is
unable to detect this binding.
Figure 4: Protease digestion of BiP mutants. 10 µg of purified wild-type and mutant BiP were either digested with 2.0 µg of proteinase K for 25 min in the presence of 0.1 mM ATP, 0.1 mM ADP, or no added nucleotide or left undigested. Proteinase K was inactivated by adding phenylmethylsulfonyl fluoride. The samples were analyzed by SDS-PAGE and detected by Coomassie Blue staining. Positions and sizes of the major proteolytic fragments are marked at the right.
ATP did not protect a 60-kDa
fragment in either the G226D or G227D mutant, which is in agreement
with our inability to demonstrate stable binding of ATP to these
mutants. However, the 44-kDa fragment was still protected in the
absence of any added nucleotide. HPLC analyses revealed that these
mutants did not contain ATP or ADP (data not shown), suggesting that
the NH-terminal ATP binding domain folds compactly even in
the absence of nucleotide and is in keeping with data obtained for
nucleotide-free WT BiP that was also analyzed (33) .
Surprisingly, ATP did not protect a 60-kDa fragment in the T37G mutant,
even though its ATP binding affinity was normal. This suggests that
T37G is unable to undergo the appropriate conformational change upon
binding to ATP that leads to changes in the protein binding domain.
Figure 5: Peptide effect on the ATPase activity of T37G and WT BiP. ATPase activity was assayed as described in the legend to Fig. 3. Peptide I or peptide C (500 µM) was included for assaying peptide-stimulated ATPase activity. All the reactions were incubated at 37 °C for 30 min. Relative units of ATPase activity were calculated.
Figure 6: Peptide binding to WT and mutant BiP and release by ATP. Upper panel, rBiP proteins were incubated with peptide C-coupled beads at 4 °C for 1 h, washed, and then either incubated with ATP (+) or without ATP(-) for 30 min at room temperature. Beads were washed and bound proteins were analyzed by SDS-PAGE. Bottom panel, the density of the protein bands in the upper panel were quantitated by scanning densitometry. The amount of bound protein without ATP represented 100% binding and the amount remaining after ATP treatment was calculated as a percent of the ATP(-) bands.
Our identification of three classes of BiP ATPase mutants further delineates the events in ATP hydrolysis and allows us to separate each event to determine its functional significance. Since the residues we mutated in BiP are absolutely conserved in the highly homologous ATP binding domain of all HSP70 members, we anticipate that similar changes in other members would produce similar classes of mutants.
Two dominant negative mutants have been identified in Kar2
(yeast BiP) that resulted from the mutation of Gly and
Gly
to aspartic acid.
Although these mutants
have not been characterized biochemically, examination of side chain
coordinate data revealed that in hsc70 the corresponding residues
(Gly
and Gly
) are only 3.66 and 4.09
Å, respectively, from the
phosphate of ATP. It is easy to
imagine that substitution of a large, negatively charged side chain at
this site would interfere with the binding of both ADP and ATP. Indeed,
we found that mutation of the corresponding residues in hamster BiP
(G226D and G227D) yielded ATP and ADP binding mutants. Although the K
values for these two mutants could not be
directly obtained, their binding affinities for ATP were significantly
lowered as estimated from their K
. Because these
mutants still have partial ATPase activity and can be labeled with
azido-ATP, we suspect that the decreased affinity is most probably due
to an increased off-rate. This correlates well with our protease
digestion data demonstrating that there was no protection of the 60-kDa
fragment that is characteristic of stable ATP binding.
However, our
inability to detect nucleotide binding for the G226D and G227D mutants
remains a concern. We checked to see if this could be a result of
aggregate formation at the high protein concentrations used for this
assay. However, we found that the proteins were completely soluble
after equilibrium dialysis and existed as both dimers (60%) and
monomers (
40%). Interestingly, the mutant dimers could not be
shifted to monomers by the addition of 200 µM ATP, whereas
the WT BiP dimers were readily converted to monomers. Therefore, under
the nucleotide binding conditions, the WT BiP was monomeric while these
two mutants were largely dimeric (data not shown). This difference in
molecular forms could further contribute to our inability to measure
stable nucleotide binding for the G226D and G227D mutants since BiP
dimers may have a lower affinity for ATP(29) . In summary, we
have been as rigorous as possible in an attempt to resolve the
discrepancy between the K
measured for these
mutants and our inability to detect stable nucleotide binding and can
find no obvious technical reason for it. Despite this, all of our
assays demonstrate that both mutants are impaired in their ability to
bind to nucleotide.
The T229G mutant was described previously as a
BiP ATPase mutant(16) . The characterization presented here
demonstrates that the defect leading to reduced ATPase activity is not
due to an inability of this mutant to bind ATP or to undergo a
conformational change after binding ATP, but is due to its inability to
hydrolyze ATP. The importance of T229 in ATP hydrolysis was anticipated
by structural predictions(13, 14) . This threonine has
been mutated in both dnaK (Thr) and hsc70
(Thr
). Characterization of the dnaK Thr
mutants revealed that substitution of an alanine, valine, or
aspartic acid at this site greatly reduced the ATPase activity of the
mutants (0.6, 3, and 7%, respectively) compared to that measured for WT
dnaK(19) . These mutants still bind ATP-agarose (19) ,
and the T199A mutant undergoes a conformational change upon ATP binding
resulting in the release of substrate protein(10) , which is
entirely consistent with our characterization of the BiP T229G mutant.
Unlike the data obtained for dnaK and BiP, mutation of the
corresponding threonine (Thr
) in hsc70 results in an
increase of both k
and K
(21) .
HSP70 proteins bind nucleotide
in the NH-terminal domain and polypeptide in the C-terminal
domain. ATP causes the release of bound polypeptides and peptide
binding can activate the ATPase activity, suggesting that communication
between the two domains is important for the functions of HSP70
proteins. The protection of a 60-kDa fragment with ATP versus a 44-kDa fragment with ADP in the presence of proteases (11, 12) implies that a conformational change takes
place in HSP70 proteins upon ATP binding that affects the protein
binding region residing between the 44- and 60-kDa cleavage
sites(30) . The T37G mutant binds ATP and ADP with normal
affinity, but a 60-kDa fragment was not protected during proteolysis in
the presence of ATP, suggesting that the ATP-induced conformational
change does not occur in the T37G mutant. This uncoupling was further
confirmed by the finding that its ATPase activity was not stimulated by
peptide. The fact that the K
for ATP and ADP were
not altered in this mutant suggests that the conformational change in
the protein binding domain does not act to stabilize ATP in the pocket,
but to transduce a signal from the ATPase domain to the protein binding
domain for substrate release. A dnaK transducing mutant was recently
described that resulted from the mutation of Glu
(Glu
in BiP) to alanine, leucine, or lysine (31) suggesting that there may be several ways to uncouple the
ATP-mediated release of proteins from HSP70 members.
A recent report demonstrated that the rate of peptide release from dnaK and hsc70 after ATP binding was faster than the rate of ATP hydrolysis (10) , implying that ATP binding, not hydrolysis, triggered the release of peptide. The dnaK T199A mutant is impaired in its ability to hydrolyze ATP, but it is able to release denatured proteins with ATP in the presence of KCl(10) . The ability of our ATP hydrolysis mutant, T229G, to be released from peptide with ATP supports this data. The ATP binding-mediated release they described requires KCl and does not occur with NaCl(10) . We previously reported that the T229G mutant was defective in peptide release using a NaCl containing buffer(16) , but this mutant is released as readily as wild-type BiP when KCl is present in the buffer. This suggests that KCl is required for the ATP-induced conformational change in HSP70 proteins. The same group (10) found that ATP analogues were unable to release bound polypeptides from dnaK or hsc70 which is in agreement with earlier reports(2, 3, 4, 5) . However, they showed that this was not because ATP hydrolysis is necessary for the release but was because the ATP analogues do not induce a conformational change in the HSP70 proteins(10) . Our characterization of the three classes of ATPase mutants allowed us to re-examine this question and to confirm their findings in several different ways. First, the ATP hydrolysis mutant (T229G), that binds ATP with wild-type affinity but does not hydrolyze it, released peptide upon ATP addition with the same apparent kinetics as wild-type protein. Second, the ATP binding mutants (G226D and G227D) were not capable of releasing bound peptide. Finally, we were able to determine that ATP binding per se is not sufficient for peptide release, but that a concurrent conformational change induced by ATP binding is also required.
Most current models on HSP70 protein binding and release portray the ADP-HSP70 complex as peptide binding competent and ATP-HSP70 as incompetent for peptide binding. In vivo these may be the only two forms of HSP70, since the binding affinity of both ATP and ADP are very high. However, our in vitro data on the two nucleotide binding mutants demonstrate that ADP binding is not a requirement for making BiP receptive to peptides. This is further supported by the fact that the protein binding domain of hsc70 (32) and BiP(23) , expressed without the ATP binding domain, are able to bind polypeptides.
In vitro systems have provided sophisticated and essential information on nucleotide binding, nucleotide-induced changes, and nucleotide hydrolysis by HSP70 proteins and delineated the requirements for in vitro release of bound proteins. These data have been instrumental in allowing investigators to produce models of how the ATP binding/hydrolysis activities of HSP70 proteins regulate their function in vivo. However, it has not been possible to directly test these models because separating these events in vivo is not feasible. Our identification of BiP ATPase mutants that are defective in the various steps in ATP hydrolysis, coupled with an in vivo expression system and a species-specific antisera to recognize the transfected mutants (23) will allow for the first time the direct testing of the various HSP70 functional models in vivo.