(Received for publication, June 20, 1995; and in revised form, September 7, 1995)
From the
HSP70 family proteins bind ATP and hydrolyze it, but the precise
role of these activities in their in vivo chaperoning function
has not been determined. In this report, we characterized wild-type
hamster BiP isolated from bacteria in terms of its ATP binding and
ATPase activities. Recombinant BiP behaved essentially the same as
endogenous BiP in terms of oligomeric status, protease digestion
patterns, and ATPase properties. By engineering a Factor Xa cleavable
site following the His tag which was used for affinity purification, we
demonstrated that the six histidines had no effect on either the
structural or ATPase properties of recombinant BiP. We also found that
bacteria-synthesized BiP had a tightly bound ADP that was resistant to
dialysis. Removal of the bound nucleotide allowed us to directly
measure the binding affinity of ATP and ADP to BiP (K of 0.2 µM for ATP and
0.29 µM for ADP) by equilibrium dialysis. Careful
characterization of wild-type BiP will allow us to use this system to
characterize BiP ATP binding site mutants that can be used to probe the
role of ATP binding and ATPase activity in BiP functions.
The heat shock protein 70 (HSP70) ()family is
comprised of a highly conserved class of molecular chaperones. Members
of this family exist in all species from Escherichia coli to
human and in all the organelles of eukaryotic
cells(1, 2) . Most of these proteins are
constitutively expressed and are further induced under conditions of
stress like heat shock or other physiological insults to the cells (1) . Immunoglobulin heavy chain binding protein (BiP) is the
eukaryotic endoplasmic reticulum (ER) member of the HSP70 family, and
is one of the major resident ER proteins. BiP is proposed to play a
role in protein folding, subunit assembly, and subsequent transport of
proteins from the ER(2) . BiP associates transiently with a
variety of nascent secretory proteins (3, 4, 5, 6, 7) and more
stably with malfolded or unassembled proteins whose transport from the
ER is
blocked(3, 4, 5, 8, 9) .
However, the extent of BiP's involvement in these processes is
not clearly understood.
All HSP70 family members possess a highly
conserved NH-terminal ATP binding domain and a more
variable COOH-terminal protein binding
domain(10, 11) . The three-dimensional structure of
the NH
-terminal 44-kDa proteolytic fragment of bovine brain
hsc70, a cytoplasmic HSP70, has been determined. This fragment contains
the ATP binding site, and its structure is very similar to that of two
other adenine nucleotide binding proteins: actin and hexokinase, even
though their amino acid sequence homology is not
high(12, 13) . Studies on both recombinant and
purified bovine hsc70 revealed that hsc70 binds ATP and ADP with K
values in the order of
10
-10
M(14, 15, 16, 17, 18) .
Although all family members presumably have a similar nucleotide
binding structure, direct binding studies have not been done on BiP or
other HSP70 members. The HSP70s also have a rather weak intrinsic
ATPase activity. For BiP, the reported turnover numbers for ATPase
activity range from 0.02 to 0.35
min
(19, 20) . Studies with normal
endogenous BiP purified from dog pancreas demonstrated that the ATPase
activity is optimal at acidic pH and low salt concentrations, requires
magnesium, and is strongly inhibited by calcium(20) . There has
been no thorough characterization of the enzymatic properties of
bacterially expressed wild-type recombinant BiP (rBiP).
In this
report, we describe the nucleotide binding properties and ATPase
activity of rBiP purified from bacteria. Hamster BiP was tagged with
six histidines at the NH terminus (6X-His), expressed, and
affinity purified on Ni
-agarose to near homogeneity.
By engineering a Factor Xa cleavage site immediately following the
histidines and comparing the 6X-His-tagged BiP with the tag-cleaved
BiP, we found that the six-histidine tag had no effect on either the
structural or ATPase properties. The ATPase activity of rBiP was
characterized in terms of pH optimum, salt, and divalent cation
requirements, and these properties were essentially the same as those
reported for BiP purified from tissue(20) . Like hsc70, the
rBiP purified from bacteria contained tightly bound ADP, and removal of
this nucleotide allowed us to measure the nucleotide binding directly
by equilibrium dialysis. These characterizations are not only important
for establishing the enzymatic parameters of BiP, but will also enable
us to identify BiP ATPase and ATP binding mutants that can be used to
determine the role of nucleotide in BiP's function.
In order to evaluate any
effects of these histidines, a specific protease cleavage site was
inserted between the 6X-His tag and the sequence corresponding to the
NH terminus of the mature BiP protein (Fig. 1A). The Factor Xa recognition tetrapeptide,
IEGR, was chosen for the following reasons: 1) this sequence does not
exist in the BiP molecule, and 2) cleavage with Xa occurs immediately
after the arginine residue in the tetrapeptide, producing a BiP
molecule identical in amino acid sequence to mature BiP. The
bacterially expressed BiP with 6X-His-IEGR on the NH
terminus was first purified on a Ni
-agarose
column and subsequently digested with Factor Xa for varying amounts of
time (Fig. 1B). The cleavage was nearly complete at 18
h. After rebinding the sample to Ni
-agarose to remove
any remaining uncleaved BiP, the Xa-cleaved protein is quite pure (Fig. 1B, purified). Isolation of pure rBiP
protein containing the 6X-His tag, as well as protein devoid of the His
tag, allowed us to determine whether the His tag affected either
structural or ATPase properties of rBiP.
Figure 1:
Engineering of a Factor Xa cleavage
site following the six-histidine tag on rBiP and purification of
tag-cleaved rBiP (mature BiP). A, NH-terminal
amino acid sequence alignment of dnaK, hsc70, and BiP. The locations of
His tag and Factor Xa recognition site on BiP constructs are underlined. The boxed sequences denote the conserved
NH
-terminal region of all HSP70 members. B,
affinity purified His-Xa-BiP was digested with Factor Xa for different
time periods and an aliquot of each was analyzed on SDS-PAGE. The 18-h
sample was reincubated with Ni
-agarose to remove the
remaining His-tagged BiP and obtain purified mature
BiP.
Figure 2: Structural analysis of His-Xa-BiP, mature BiP, and His-BiP. A, 10 µg of purified recombinant His-Xa-BiP, Xa cleaved BiP (mature), and 6X-His-tagged BiP (His-BiP) were electrophoresed under nondenaturing conditions. The proteins were visualized by Coomassie Blue staining. B, 10 µg of the three purified rBiP preparations were either digested with 2.0 µg of proteinase K in the presence of 0.1 mM ATP, 0.1 mM ADP, or no added nucleotide, or left undigested. 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.
Partial protease digestion is
often used as a measure of the structural integrity of a protein. The
HSP70 proteins produce very distinctive proteolytic patterns when
digested in the presence of nucleotides. ATP protects 60- and 44-kDa
fragments, whereas, ADP protects only the 44-kDa
fragment(11, 20) . In order to check the structural
integrity of our three forms of rBiP, we digested the various samples
with proteinase K, a nonspecific serine protease, in the presence of
ATP or ADP. For all three of the BiP preparations, both a 60- and a
44-kDa fragment were protected in the presence of ATP, and a 44-kDa
fragment was protected when ADP or no nucleotide was present (Fig. 2B). These protected fragments were all derived
from the NH-terminal domain, because they could still bind
to Ni
-agarose (His-Xa-BiP and His-BiP) but could not
be immunoprecipitated with a COOH-terminal specific antiserum (data not
shown). The observation that samples with no nucleotide added produced
the same digestion pattern as samples to which ADP was added implies
that either the rBiP already contained bound ADP or that the
NH
-terminal domain folds very compactly even without
nucleotide. Together, these data suggest that bacterially produced rBiP
proteins retained the structural integrity of the native BiP protein,
and that the presence of a histidine tag did not grossly alter this
structure.
Figure 3:
Characterizing the ATPase activity of
recombinant BiP preparations. A, the ATPase activity of all
three rBiP preparations and bovine serum albumin were assayed under
standard conditions as described under ``Materials and
Methods'' using 2.0 µM rBiP and 1.0 mM [-
P]ATP. Aliquots were removed at 0,
10, 20, and 30 min and nanomoles of ATP hydrolyzed were calculated
basing on scintillation counts and specific activity. B, pH
effect on the ATPase activity of tag-cleaved rBiP (mature BiP)
and 6X-His-tagged rBiP (His-BiP) were measured using different
buffers (``Materials and Methods'') but keeping all other
conditions the same. The relative units of ATP hydrolysis were simply
derived from the cpm at 30 min. C, the effects of KCl (
)
and NaCl (
) on the ATPase activity of mature BiP were analyzed
by including different amounts of either KCl or NaCl (0-600
mM) in the assay mixtures. Relative units of ATP hydrolysis
were determined as in B. D, the effects of MgCl
(
), Mg(CH
COO)
(
), and
CaCl
(
) on the ATPase activity of mature BiP were
determined by adding the divalent cations to 0.1, 1.0, 10, or 100
mM to the reaction using the standard buffer but devoid of
MgCl
. Again relative units of ATP hydrolysis were
determined as in B. The salt effect and divalent cation effect
were indistinguishable for mature BiP and His-BiP and thus for
simplicity, only the data for mature BiP is
displayed.
The pH effect on BiP's ATPase activity was examined by using the same buffers described for assaying endogenous BiP(20) . Peak ATPase activity was obtained at pH 5.0 while either increasing or decreasing the pH from pH 5.0 significantly lowered the ATPase activity (Fig. 3B). For all further ATPase determinations, pH 7.0 was used because: 1) the ER pH is estimated to be approximately pH 7.0; 2) pH 7.0 is in a region of the pH curve with a shallow slope, thus minimizing the potential for large measurement errors; and 3) the ATPase and ATP binding activities of hsc70 were analyzed at pH 7.0(14, 18, 30) . Thus, by performing our ATPase assays at pH 7.0, it will be possible to compare findings between these two related proteins.
We next assayed the effects of
salt and divalent cations on BiP's ATPase activity. The data for
cleaved and His-tagged BiP were indistinguishable, and thus, for
simplicity, only that for cleaved BiP (mature) is presented. The
optimal salt concentration was about 25 mM for both NaCl and
KCl, but at all concentrations measured, KCl produced a higher ATPase
activity than NaCl (Fig. 3C). Therefore, all remaining
assays were performed with 25 mM KCl. Calcium was strongly
inhibitory to BiP's ATPase activity, and magnesium was required
for the optimal enzyme activity (Fig. 3D). This is
consistent with the requirements for both native BiP and
hsc70(20, 30) , but unlike data on hsc70,
Mg(CHCO)
was not preferred over
MgCl
. The optimal concentration of MgCl
required for rBiP's ATPase activity ranged from between 1
and 10 mM. The ATPase activity of our rBiP could be stimulated
2-3-fold by peptides (see (43) , accompanying article),
which is similar to data for native mammalian BiP and
hsc70(28, 31) . Taken together, these data demonstrate
that the ATPase properties of our rBiP purified from bacteria are
extremely similar to those of BiP purified from dog
pancreas(20) . In no case was there a significant difference
between 6X-His-tagged and tag-cleaved BiP, demonstrating that the
6X-His tag does not affect any of the various aspects of BiP's
ATPase activity. In the following study(43) , 6X-His BiP was
used in order to avoid additional manipulations to the recombinant
proteins during purification.
Figure 4: HPLC analysis of nucleotide bound to rBiP. a, buffer alone was extracted as a control and the background profile was determined. b, nucleotide was extracted from 10 µM rBiP as described under ``Materials and Methods.'' c, rBiP was preincubated with 600 molar excess of AMPPNP to replace bound nucleotide and then extensively dialyzed to remove both bound and free nucleotides. Nucleotide was extracted as in b. A control sample containing a mixture of ADP, AMPPNP, and ATP was injected to determine where each of them eluted and each is denoted with a downward arrow.
At this point it was necessary to prepare nucleotide-free BiP in order to measure the nucleotide binding constants for ATP and ADP. We used the method developed for preparing nucleotide-free hsc70(18) . A 600-fold molar excess of AMPPNP was added to rBiP, the sample was incubated at room temperature for 1 h to replace the bound ADP, and then the bound AMPPNP, as well as free nucleotides, were removed by dialysis. Over 95% of the bound ADP was removed after this treatment, as shown by HPLC analysis (Fig. 4). The nucleotide-free BiP was stable for about 2 weeks when kept at 4 °C as determined by measuring ATPase activity (not shown). The oligomerization state and protease digestion patterns were not altered by removing the nucleotide, suggesting that ADP is not required for BiP oligomerization and that nucleotide-free BiP is structurally similar to BiP containing bound ADP (not shown).
Figure 5: Kinetic analysis of BiP's ATPase activity. A, enzyme (BiP) concentration dependence of BiP's ATPase activity was determined using 200 µM ATP at pH 7.0 and 37 °C for 30 min. Relative units of ATP hydrolysis were directly simplified from cpm. B, the time dependence of BiP's ATPase activity was assayed using 0.05 µM BiP and 0.5 µM ATP. Bovine serum albumin (0.05 µM) was assayed as a control for both background ATP hydrolysis and organic extraction. C, ATP concentration dependence of BiP ATPase activity was determined using 0.05 µM BiP (nucleotide-free) and 0.5-6.0 µM ATP for 30 min at 37 °C. D, double reciprocal plot was derived from C using Clelend's kinetic software.
Figure 6:
Binding of ATP and ADP to nucleotide-free
rBiP. The binding of ADP (A) and ATP (B) to
nucleotide-free rBiP was determined by equilibrium dialysis. Varying
amounts of [C]ATP or ADP (abscissa)
with constant specific activity were added to both sides of the
dialysis membrane. The amount of labeled nucleotide associated with the
protein at equilbrium is plotted on the ordinant. The data was
analyzed in a Scatchard plot (inset) using the calculated amount of
bound and free nucleotide.
The ability to identify and fully characterize BiP ATP binding and ATP hydrolysis mutants requires both a knowledge of the normal kinetic values for wild-type BiP, and a method to isolate mutant proteins that does not require ATP binding or that does not result in co-contamination with wild-type BiP protein. Tagging hamster BiP with six histidines at its amino terminus allowed easy purification of the rBiP from bacteria, but unlike native BiP, resulted in the addition of non-native amino acids to the ATP binding domain that could affect kinetic measurements. To determine if the 6X-His tag affected BiP, we engineered a factor Xa cleavage site immediately downstream of the 6X-His tag so it could be removed, producing a protein that was identical in sequence to mature hamster BiP isolated from cells.
The 6X-His tagged and the tag-cleaved rBiP seemed to be structurally identical to native mammalian BiP as judged by protease protection assays (20) and by their ability to form dimers and monomers(25, 28) . Similar to other studies performed on native BiP(29, 31) , we found that the dimers were converted to monomers by peptide binding (not shown), and that the ATPase activity was stimulated approximately 2-3-fold by peptide ((43) , accompanying paper). This is in contrast to another report, in which rBiP was isolated, in part, on ATP-agarose columns(29) . Their rBiP was primarily monomeric and was not stimulated by peptide. We found that all three forms of our rBiP had the same rates of ATP hydrolysis, and that their pH and salt optima, as well as the divalent cation requirements for this activity were the same as those reported for native BiP(20) . Thus, our purification method produced rBiP proteins that appeared to be structurally very similar to native BiP, and the 6X-His tag did not apparently affect structural or enzymatic parameters.
Our
recombinant BiP possessed tightly bound ADP, just like native hsc70
isolated from bovine brain(14, 27) . Unlike the
purification scheme for bovine hsc70(14, 27) , we did
not use nucleotide columns and ATP elution in our isolation. Therefore,
the bound ADP must come from the bacteria and is most likely a normal
component of HSP70 proteins. After producing nucleotide-free BiP, we
performed ATPase assays to determine the K (1.48
± 0.1 µM) and V
(5.12
pmol/min/µg) of wild-type BiP. The variations between reported
values for V
of HSP70 proteins are surprisingly
large. The V
values for BiP range from 0.2 to
4.7(19, 20) , for hsc70 from 1.2 to
12(32, 33) , and for dnaK from 2.5 to 600
pmol/min/µg(34, 35) . This variation could stem
from the fact that, in most studies, the percent of the preparation
that was active is not known, no attempt was made to remove bound
nucleotide before performing the assays, and finally, the purity of the
various preparations used in these studies is not known. Because the
HSP70s are very poor ATPases, even a small amount of contamination with
a much stronger ATPase could have profound effects on the V
value. We feel that our determination of V
and K
values are
reliable, because we ensured that our protein possessed nearly full ATP
binding activity and was free of nucleotide before performing kinetic
studies. We determined that our preparation was free of contamination
by immunoblotting with anti-dnaK antisera and by performing in
vitro kinase assays (data not shown). The purity of our rBiP
preparation was further corroborated by our isolation of an
ATPase
mutant, T229G BiP (43) that was
purified in the same way. Finally, care was taken to ensure that our
studies followed strict enzymatic criteria.
There is currently no
reported data on direct nucleotide binding for BiP, however, there are
reports for hsc70 prepared either from bovine brain or bacteria with K values for ADP or ATP ranging from
10
to 10
M. Using
initial rate binding, Schmid et al.(14) measured
nucleotide binding activities of bovine hsc70 and reported a K
of 1.35 µM for Mg-ADP and a K
of 0.7 µM for Mg-ATP, and found
that there was 0.4 mol of nucleotide/mol of protein that was not
removed by dialysis. Using equilibrium dialysis on hsc70, Palleros et al.(15) measured a K
of 1.6
µM for Mg-ADP and a K
of 9.5
µM for Mg-ATP(15) . These weaker binding
affinities, as compared to other reported values may be due to the
presence of bound nucleotides in the preparations which could distort
the binding data. Using nucleotide-free recombinant hsc70, Wang and Lee (16) reported a K
of 0.2-0.3
µM for Mg-ATP (16) , and Ha and McKay (17) reported a K
of 0.042 for Mg-ATP and
0.11 µM for Mg-ADP. The values for recombinant hsc70 are
close to our measurements for recombinant BiP. Using AMPPNP as a
primary binder in a competition study, Gao et al.(18) measured a K
of 0.012 µM for Mg-ATP and 0.018 µM for Mg-ADP for
nucleotide-free hsc70 purified from bovine brain. These values are
somewhat lower than those for recombinant hsc70 or BiP, and the
discrepancy may result either from differences in the experimental
methods used, or from an intrinsic difference between native and
recombinant proteins.
The weak ATPase activity and high ATP binding affinity of BiP measured in vitro suggests one of two things. First, other ATPase activating proteins may exist. In bacteria, two heat shock proteins, dnaJ and grpE, stimulate the ATPase activity of dnaK up to 50-fold (36) . Cytosolic homologues of dnaJ have been identified in human and yeast cells(37, 38, 39, 40) , and an ER protein with homology to dnaJ has been isolated in yeast(41) . However, no data are available to demonstrate that these proteins modulated the ATPase activity of eukaryotic HSP70 proteins. Alternatively, ATP binding may be more important to the function of the HSP70 proteins than ATP hydrolysis. This may be particularly true for BiP since the assumed ER conditions, pH 7.4 and millimolar calcium, strongly inhibit BiP's ATPase activity. An in vitro study using purified proteins demonstrated that ATP binding, but not ATP hydrolysis, was required for the release of bound polypeptides from hsp70 and dnaK(42, 43) .
In summary, we have
isolated recombinant BiP protein from bacteria and demonstrated that
enzymatically it behaves very much like native BiP isolated from
mammalian cells. Using rBiP purified by a single step, we determined
the K and V
values for
wild-type BiP and provided the first determination of nucleotide
binding affinities for BiP. The characterization of wild-type BiP
activity permits us to analyze the various BiP ATPase mutants (43) to determine whether they represent nucleotide binding or
hydrolysis mutants. This should then allow us to distinguish the role
of ATP binding from that of ATP hydrolysis in BiP function.