(Received for publication, July 16, 1996, and in revised form, December 6, 1996)
From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette Cedex, France
We have previously shown that the molecular
chaperone HSC70 self-associates in solution into dimers, trimers,
and probably high order oligomers, according to a slow temperature- and
concentration-dependent equilibrium that is shifted toward
the monomer upon binding of ATP peptides or unfolded proteins. To
determine the structural basis of HSC70 self-association, the
oligomerization properties of the isolated amino- and carboxyl-terminal
domains of this protein have been analyzed by gel
electrophoresis, size exclusion chromatography, and analytical
ultracentrifugation. Whereas the amino-terminal ATPase domain (residues
1-384) was found to be monomeric in solution even at high
concentrations, the carboxyl-terminal peptide binding domain (residues
385-646) exists as a slow temperature- and
concentration-dependent equilibrium involving monomers,
dimers, and trimers. The association equilibrium constant obtained for
this domain alone is on the order of 105
M1, very close to that determined previously
for the entire protein, suggesting that self-association of HSC70 is
determined solely by its carboxyl-terminal domain. Furthermore,
oligomerization of the isolated carboxyl-terminal peptide binding
domain is, like that of the entire protein, reversed by peptide
binding, indicating that self-association of the protein may be
mediated by the peptide binding site and, as such, should play a role
in the regulation of HSC70 chaperone function. A general model for
self-association of HSP70 is proposed in which the protein is in
equilibrium between two states differing by the conformation of their
carboxyl-terminal domain and their self-association properties.
Members of the highly conserved 70-kDa heat shock protein family (HSP70)1 are involved in several cellular processes such as protein folding, assembly and disassembly of multimeric proteins, protein translocation across membranes, protein degradation, and signal transduction (for reviews see Refs. 1-4). They are thought to act as molecular chaperones by transiently binding hydrophobic regions exposed to the solvent in the nonnative conformations of proteins, thereby preventing off-pathway reactions that lead to aggregation (5).
A prominent member of this family, the mammalian, constitutively expressed, 70-kDa heat shock cognate protein (HSC70), has been shown to bind peptides and unfolded proteins (6-8) and to possess refolding activity in the presence of the cochaperone DnaJ (9, 10). HSC70 shows a very weak ATPase activity that can be stimulated 2-5-fold upon binding of peptides, unfolded proteins, clathrin light chains, and cochaperones of the DnaJ family (11-14). HSC70 seems to function through cycles of binding and release of polypeptide substrates coupled to binding and hydrolysis of ATP (15), in a mechanism involving cochaperones of the DnaJ protein family and a newly isolated factor, Hip (16).
HSC70 is made of two domains, an NH2-terminal domain of 44 kDa (residues 1-384), which binds and hydrolyzes ATP, and a COOH-terminal domain of about 30 kDa (residues 385-646), which contains the peptide binding site (17, 18). The three-dimensional structure of the NH2-terminal ATPase domain has been solved by crystallography (19), and the secondary structure topology of the peptide binding site (residues 385-543) has been determined by NMR methods (20). Recently, the structure of a complex between a seven-residue peptide and the COOH-terminal domain of DnaK, the bacterial HSP70, has been established (21). However, the three-dimensional structure of the entire protein is still unknown.
Self-association is a general and well conserved feature of the HSP70 protein family. BiP (22-24), the constitutive HSC70 (6, 25-30), the heat shock-inducible HSP70 (31), plant HSP70 (32), and DnaK (33), all show self-association properties. Nevertheless, the structural basis and molecular mechanism of such properties have remained undocumented.
To address these questions and define the way self-association of HSC70 may relate to its function, we started the investigation of this process by studying the structure of HSC70 in solution. Using a wide range of biophysical and biochemical techniques, we showed that HSC70 self-associates in solution, in a reversible fashion, into dimers, trimers, and probably high order oligomers, with a dissociation constant of about 5-10 µM (34). Next, we analyzed the effects of the natural substrates on this equilibrium, and showed that whereas in the presence of ADP, HSC70 exists as a slow and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, peptides, or unfolded proteins, this equilibrium is shifted toward the monomer (35). Most importantly, and since the dissociation process of HSC70 oligomers into monomers appeared to be very similar to that of unfolded protein from HSC70, occurring upon ATP binding but not ATP hydrolysis, we proposed that binding of HSC70 to itself may occur via the peptide binding site and may mimic the binding of HSC70 to unfolded protein substrates (35).
To test this hypothesis, we undertook in the present work the identification of the oligomerization domain and analyzed the self-association properties of two mutants corresponding to the NH2- and COOH-terminal domains of HSC70.
Restriction endonucleases were from New England Biolabs, and T4 DNA ligase was from Pharmacia Biotech Inc. DNA sequencing was accomplished using the T7 sequencing kit from Pharmacia. PET 14b vector and Ni2+-agarose were purchased from Tebu-Novagen. FPLC products were from Pharmacia, and all other chemicals were from Merck.
Construction of the Plasmids Expressing HSC70 NH2-terminal and COOH-terminal DomainsThe plasmid carrying the coding sequence of HSC70 NH2-terminal domain was designed as follows. Site-directed mutagenesis was used to introduce an NdeI site at the start codon of the HSC70 coding sequence that had been previously cloned in the PstI sites of the pUC119 polylinker. Then a Pac1 site was introduced to create a stop codon at codon 385, resulting in the replacement of Asp383 and Lys384 by a valine and an asparagine, respectively. The resulting NdeI-PacI fragment was then cloned between the corresponding sites of the pNB5 expression vector described previously (30). The final plasmid, called pFB5, allowed the expression of the NH2-terminal domain of HSC70 (residues 1-384) in Escherichia coli.
The HSC70 COOH-terminal domain (385-646) was expressed using the T7 expression system of E. coli. Site-directed mutagenesis was used to introduce an NdeI site at codon 383 in the HSC70 coding sequence of the pFB7 plasmid described previously (30), thus leading to the replacement of codon 384 by a start codon. Then, after removal of the NdeI-NdeI fragment (codons 1-383), the plasmid was ligated on itself, giving rise to the pNB28 plasmid. Finally, the NdeI-BamHI fragment of pNB28 was cloned into the NdeI-BamHI sites of pET14b vector, resulting in the NH2-terminal fusion of six histidine residues to the HSC70 COOH-terminal domain (residues 385-646). The integrity of all constructions described above was verified by nucleotide sequencing (36).
Protein Expression and PurificationRecombinant HSC70 was
expressed and purified as described previously (30, 34). The
recombinant NH2-terminal domain of HSC70 was expressed and
purified as described for HSC70 except that the first ion exchange
chromatographic step was replaced by a 60% ammonium sulfate
precipitation. After centrifugation, the ammonium sulfate concentration
of the supernatant was adjusted to 80%, and the sample was submitted
to centrifugation. The resulting pellet was resuspended in 20 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM -mercaptoethanol, and 3 mM
MgCl2, dialyzed in the same buffer, and applied onto an
ATP-agarose affinity column as described previously (30, 34).
The HSC70 (His)6-COOH-terminal domain was expressed and
then purified using His-bind resin (Ni2+-agarose),
according to the recommendations of the manufacturer. The eluted
protein was subjected to a thrombin digestion to remove the histidine
tail, and any uncleaved His-COOH-terminal domain was removed by
submitting the whole sample to a chromatography on an
Ni2+-agarose column. After elution, the protein was
concentrated and stored at 80 °C as described previously (30). The
protein concentration was determined by the Lowry method (55) using
bovine serum albumin as a standard, and all protein concentrations
given in the tables and figures are based on the molecular mass of the
monomer. Activity of the purified proteins was checked by the measure
of the ATPase activity as well as polypeptide substrate binding.
Polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (SDS) was carried out in 0.75-mm-thick 12% acrylamide slab gel according to Laemmli (37). Gel electrophoresis in native conditions was performed either on a 6 or 10% acrylamide slab gel according to Kim et al. (29).
Size Exclusion ChromatographyFPLC chromatography was
carried out at room temperature on a Superose 12 column equilibrated
with 20 mM Tris-HCl, pH 7.5, 100 mM KCl, and 1 mM -mercaptoethanol as described previously (34).
Sedimentation velocity experiments were performed at 20 °C on a Beckman Optima XL-A analytical ultracentrifuge equipped with a An Ti 60 titanium four-hole rotor with two-channel 12-mm path length centerpieces as described previously (34). Data analysis was performed using the computer program SVEDBERG (38) supplied by John Philo. The apparent molecular mass was determined from the sedimentation coefficient, using the relation (S1/S2)3 = (M1/M2)2 and bovine serum albumin as a reference protein of known molecular mass and sedimentation coefficient (34, 35, 39).
Sedimentation EquilibriumSedimentation equilibrium experiments were carried out at 4 °C using three different loading concentrations and three (for the entire protein) or two (for the isolated fragments) rotor speeds. Radial scans of absorbance at 280 nm were taken at 2-h intervals, and samples were judged to be at equilibrium by the absence of systematic deviations in overlaid successive scans and when a constant average molecular weight was obtained in plots representing the average molecular weight versus centrifugation time. Sedimentation equilibrium data were analyzed using the appropriate functions by nonlinear least squares procedures provided in the Beckman Optima XL-A software package.
For data analysis according to discrete self-association models, the following general equation was used.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Average weight molecular weights
(w) were obtained, for several
rotor speeds and protein concentrations, by fitting the equilibrium
sedimentation data to a single species using the equations above. The
variation of
w as a function of protein concentration from single runs was determined by calculating the
w from
dln(C)/dr2 = M1(1
)
2/ 2RT data, on a
point by point basis, using a window of 20 points that moves through
the entire data point range.
Data analysis, according to an unlimited isodesmic association model
(40, 41), in which the equilibrium constants for the addition of
monomer to any oligomer are equal, was performed using the SEDPROG
software package provided by Greg Ralston (Ref. 42 and references
therein) and the function
(r).
(r) (43) is defined as follows,
![]() |
(Eq. 3) |
A monomer molecular mass of 29,046 Da and a partial specific volume of 0.726 ml/mg at 4 °C, calculated from amino acid composition, were used for the COOH-terminal domain. For the NH2-terminal domain and HSC70, monomer molecular masses of 42,002 and 70,870 Da and partial specific volumes of 0.733 and 0.729 ml/mg at 4 °C were used. The solvent density was taken as 1.00 g/ml.
Analysis by Polyacrylamide Gel Electrophoresis
Although a purified preparation of HSC70 appears homogeneous on a
denaturating polyacrylamide gel (Fig. 1A,
lane 2), it presents a polydispersity on a nondenaturating
gel, and at least three species are observed (Fig. 1B,
lane 1). By contrast, the NH2-terminal ATPase
domain migrates as a single band whether in native (Fig. 1B,
lane 2) or denaturing conditions (Fig. 1A,
lane 3), suggesting that it exists as a single species.
However, the COOH-terminal domain presents an electrophoretic behavior
similar to that of the entire protein. It exhibits multiple bands in
native conditions corresponding to at least three species (Fig.
1B, lane 3), although it shows a single band in
denaturing conditions (Fig. 1A, lane 4),
indicative of self-association.
Analysis by Size Exclusion Chromatography
To obtain further information about the nature and the relative
distribution of the species present in each protein preparation, HSC70
as well as the isolated domains were analyzed by size exclusion chromatography. While the NH2-terminal domain elutes as a
single sharp and symmetrical peak corresponding to a single species
having an apparent molecular mass of about 36 kDa (Fig.
2B), the COOH-terminal domain of HSC70 elutes
in two overlapping peaks, a major and broad one corresponding to
species having an apparent molecular mass of about 200 kDa and a minor
one corresponding to species having an apparent molecular mass of about
40 kDa (Fig. 2C), indicating the presence of monomeric and
various oligomeric species, probably dimers and trimers. Thus, the
COOH-terminal domain alone seems to self-associate in a manner similar
to that of the entire protein. The partial separation of these species
during chromatography is indicative of the presence of either a mixture
or a slow equilibrium between species, comparable with that described
for the whole protein (Fig. 2, A and C; Refs. 34
and 35).
To know whether the monomeric and oligomeric species of the
COOH-terminal domain exist in a noninterconvertible mixture or in a
slow equilibrium, we studied the concentration and temperature dependence of self-association for the isolated domain. As shown in
Fig. 3, progressive dilution of the COOH-terminal domain
leads to an increase in the amount of the monomeric species at the
expense of the oligomeric species (Fig. 3, D, E,
and F) in a way similar to that observed for the entire
protein (Fig. 3, A, B, and C). These
results indicate that the COOH-terminal domain, like the entire HSC70,
exists as a slow and concentration-dependent equilibrium between oligomeric and monomeric species (see also Ref. 34). This is
confirmed by the temperature dependence of the COOH-terminal domain
self-association. In fact, varying the temperature in the gel
filtration experiments had two purposes: first, to confirm that the
multiple species observed exist as a slow equilibrium and not as a
mixture, and second, to know whether the interactions involved in
stabilizing the multimeric species are of a comparable nature in the
whole protein and its COOH-terminal domain. As shown in Fig.
4, for HSC70 as well as its COOH-terminal domain,
increasing the temperature leads to the dissociation of the oligomers
into monomers. However, the COOH-terminal domain appears to be more stable at high temperatures than the entire protein, since above 42 °C it is still undergoing dissociation into monomers (Fig. 4,
G and H), whereas HSC70 becomes aggregated as
indicated by the presence of a single peak eluting with the void volume
of the column (Fig. 4, C and D), due presumably
to heat-induced denaturation and subsequent aggregation. Even at higher
temperatures, up to 60 °C, the COOH-terminal domain does not seem to
aggregate and rather elutes as a single chromatographic peak
corresponding to monomeric species (data not shown).
It is not clear whether increasing temperatures alter the equilibrium distribution of the species, and thus their respective thermodynamic stability, or the rate of conversion between species, although an effect on the rate has been suggested (46). In the absence of calorimetric studies and thermodynamic parameters, interpretation of these results in terms of the nature of forces contributing to stability could only be speculative. Thus, it could be concluded that whether in the case of the COOH-terminal domain or HSC70, the monomer-oligomer equilibrium is temperature-dependent and that the interactions involved in stabilizing their multimeric species are probably of a similar nature, suggesting that self-association of HSC70 could be accounted for by the COOH-terminal domain.
Analysis by Analytical Ultracentrifugation
Sedimentation VelocityResults of sedimentation velocity
experiments are summarized in Table I. An average
sedimentation coefficient, 20,w, of 3.46 S is obtained for the NH2-terminal domain. This
value corresponds to an apparent molecular mass of 45,240 Da, close to
the molecular mass of the monomer predicted from the amino acid
sequence (42,002 Da). Increasing protein concentrations, up to 50 µM, did not significantly affect this value, and the NH2-terminal domain remained monomeric over all of the
concentration range (data not shown). Moreover, extrapolating the
20,w to 0 gives an
20,w0 of 3.24 S, which
corresponds to a molecular mass value of 41,108 Da, even closer to that
of the predicted one.
|
By contrast, the average sedimentation coefficients determined for the
entire HSC70 and the COOH-terminal domain, 6.67 and 4.25 S,
respectively, correspond to molecular masses of 121,300 and 61,700 Da,
respectively. These values are higher than those predicted from the
amino acid sequence for the monomer (70,870 Da for HSC70 and 29,046 Da
for the COOH-terminal domain) and reflect the self-associating nature
of these proteins. As described previously, the best fit of the
sedimentation velocity data of HSC70 was obtained using a
monomer-dimer-trimer model (34). Since the COOH-terminal domain seemed
to behave like HSC70 in terms of self-association, a three-component
model system has been used to fit the sedimentation velocity data. The
s20,w values of 2.91, 4.53, and 6.76 S
returned by the fitting procedure correspond to molecular masses of
34,900, 67,800, and 119,900 Da, respectively, and are compatible with a
monomer, a dimer, and a trimer. Similar fitting of HSC70 data gave
values of 4.63, 7.09, and 9.97 S, which are close to 20,w0 values of HSC70
monomer, dimer, and trimer published previously (34). The fact that
each species of the equilibrium could be separated from the others and
characterized by a distinct sedimentation coefficient, as if all
species coexisted in a noninterconverting mixture, is indicative of a
slowly equilibrating system as compared with the time of
sedimentation.
Together, these results are in agreement with the chromatography data and confirm that, while the NH2-terminal domain is monomeric in solution, even at high concentrations, the COOH-terminal domain self-associates, in a manner similar to that observed for the entire protein, giving rise to dimers and trimers in a relatively slow concentration- and temperature-dependent equilibrium.
Sedimentation EquilibriumThe weight average molecular weight
of the entire protein and the isolated NH2- and
COOH-terminal domains at similar concentrations (about 8.5 µM) were determined by fitting the equilibrium
sedimentation data to a single species. Whereas the weight average
molecular weight of the NH2-terminal domain was found to be
40,000, close to the molecular mass of the monomer obtained from the
amino acid composition (42,002 Da), that of the entire protein
(127,000) and that of the COOH-terminal domain (65,000) are much higher than expected (70,870 and 29,046, respectively, suggesting
self-association of these proteins) (data not shown). This is confirmed
by measuring the variation of the weight average molecular weight as a
function of protein concentration (Fig. 5). While the
weight average molecular weight of the NH2-terminal domain
is independent of concentration and is about 42,000, that of the
COOH-terminal domain increases with concentration and corresponds to
the molecular mass of monomeric species (29,046 Da) at the lowest
concentration and dimeric species (about 58,092 Da) at the highest
concentration (Fig. 5B). This behavior is quite similar to
that of entire protein for which the weight average molecular weight is
close to the molecular mass of the monomer (70,870 Da) at low protein
concentrations and approaches that of the dimer (141,740 Da) at high
protein concentrations (Fig. 5A). Together, these results
indicate that the COOH-terminal domain alone accounts for the variation
of the weight average molecular weight of the protein as a whole. Since the weight average molecular weight values do not reach a plateau at
high protein concentrations, self-association goes beyond the formation
of dimers for both HSC70 and its COOH-terminal domain.
To obtain the thermodynamic parameters for the COOH-terminal domain
association equilibrium, six data sets, obtained using three initial
loading concentrations and two rotor speeds, were simultaneously fitted
to a monomer-dimer-trimer self-association model. This model has been
successfully used to fit the sedimentation equilibrium data for HSC70
in previous work (34). The choice of this model in the present work is
based on the similarity of the self-association properties of the
COOH-terminal domain and HSC70 and the nature of the oligomeric species
detected by size exclusion chromatography and analytical
ultracentrifugation. As shown in Table II, the
association constant for adding a monomer to a monomer or a monomer to
a dimer (KM-D and KD-T,
respectively) are on the order of 105
M1 (0.4 × 105 and 6.2 × 105 M
1, respectively) for the
isolated COOH-terminal domain. These constants are similar to those
determined for the entire protein (1.1 × 105 and
0.9 × 105 M
1) using the
same self-association model. By contrast, the data corresponding to the
NH2-terminal domain fit very well to a single ideal species
model, giving a molecular mass of 42,000 Da, and significantly less
well to a monomer-dimer model, giving a dissociation constant in the
millimolar range (data not shown), confirming that this domain does not
self-associate in solution. Since we showed previously that the
sedimentation equilibrium data for HSC70 could also fit to an unlimited
association model involving a single association constant for all steps
of about 105 M
1 (34), the data
for the COOH-terminal domain were fitted to the same model. As shown in
Table II, an isodesmic, unlimited association model describes the data
almost equally well as indicated by the root mean square of the fit. An
association constant of the same order of magnitude (105
M
1) is obtained, thus indicating that the
COOH-terminal domain alone is sufficient to account for the
thermodynamic properties of the protein as a whole.
|
Involvement of the Peptide Binding Site in Self-association of the COOH-terminal Domain
The binding of peptides or permanently unfolded proteins to HSC70
or DnaK promotes the dissociation of oligomers into monomers (35, 46,
47), suggesting that oligomerization of the protein is mediated by the
peptide binding site (35). As shown in Fig. 6, the
addition of peptide C, a 13-residue peptide from vesicular stomatitis
virus glycoprotein known to bind to HSC70 with a dissociation constant
of about 5-10 µM (12, 48), whether to the COOH-terminal peptide binding domain alone (Fig. 6B) or to the entire
protein (Fig. 6A), promotes the dissociation of oligomeric
species into monomers. The low solubility of peptide C did not allow
the use of higher concentrations to obtain complete dissociation of
oligomers. However, below this limit, dissociation into monomers
increases with peptide concentrations (data not shown). Altogether,
these results suggest that by binding to the peptide-binding site,
peptide C is able to shift the monomer-oligomer equilibrium toward the monomer, likely by a competition with HSC70 for the same site.
The results of this investigation indicate that all of the
self-association properties of HSC70 can be accounted for by the COOH-terminal domain only, the NH2-terminal domain not
taking part in the process. Indeed, association between HSC70 molecules through direct interactions involving their respective
NH2-terminal domains can be ruled out in view of the fact
that, even at high concentration, the isolated NH2-terminal
ATPase domain remains monomeric and behaves as a single ideal species.
Although the possibility of self-association involving the
COOH-terminal domain of one molecule and the NH2-terminal
domain of another by a head to tail mechanism cannot be excluded, this
type of association seems unlikely, since the COOH-terminal domain
alone is sufficient to account for the self-association properties of
the whole protein, not only qualitatively but also quantitatively in
terms of association mechanism and equilibrium constants. Both the
entire HSC70 and the isolated COOH-terminal domain exist as slow
temperature- and concentration-dependent equilibriums
involving monomeric, dimeric, and trimeric species characterized by
dissociation constants of the same order of magnitude, in the
micromolar range. Moreover, dissociation of the oligomeric forms of the
COOH-terminal domain occurs upon peptide binding in a manner similar to
that observed for HSC70 oligomers, suggesting that
destabilization of HSC70 oligomers is due to the disruption of contacts
between COOH-terminal domains upon peptide binding. Thus, association
of the protein to itself seems to occur exclusively via its
COOH-terminal part (see Fig. 7).
Although the COOH-terminal domain appears to be necessary and sufficient to account for the self-association properties of the entire protein, the oligomerization site within this domain has not been located. However, because peptides as well as unfolded proteins promote the dissociation of oligomers and stabilize the monomer, presumably by competing with the protein itself for the peptide binding site, self-association of the protein via the peptide binding site appears to be the most straightforward explanation. The peptide binding site of an HSC70 protomer could recognize a target site in another HSC70 protomer, as if this site were a peptide in an extended conformation or an unfolded protein, and lead to self-association (Fig. 7). This is corroborated by the fact that dissociation of HSC70 oligomers occurs after ATP binding but not hydrolysis, just as peptide and unfolded protein substrates bound to HSC70 are released upon ATP binding and not hydrolysis (49). Thus, interactions of the protein with itself should be similar to those of the protein with an unfolded polypeptide substrate.
Although the self-association properties of the protein were previously analyzed by a simple scheme in which HSC70 exists as a slow equilibrium between a monomeric form and an oligomeric form (35), the fact that two monomeric structures, differing in terms of their conformation, were characterized by small angle x-ray scattering (47, 50) and fluorescence (51), suggest that two monomeric states are accessible to the protein. Most importantly, these states correspond either to the ADP-bound form or to the ATP-bound form of the protein, which also are known to differ in terms of their self-association properties, the former being able to oligomerize, whereas the latter is stabilized as a monomer (34, 35). Thus, since the ATP-bound form is stabilized as a monomer, a conformational change appears to be necessary to give rise to an alternative monomeric structure that would be able to self-associate. The question is then whether these two states exist in an equilibrium prior to the binding of nucleotides, the effect of nucleotides being to stabilize one state or the other, or only one monomeric state exists (ADP-bound or nucleotide-free), the other one being induced by ATP binding. We favor the first hypothesis, since it is possible now to obtain, by mutagenesis, monomeric states unable to oligomerize and others that are able to do so in the absence of nucleotides.2 Based on these considerations, if we assume that these two monomeric structures exist in an equilibrium in the absence of nucleotide, the following model can be proposed. In this model, the protein is seen as a slow equilibrium between two monomeric states, state I and state II, differing by the conformation of the COOH-terminal domain and particularly by that of the peptide binding site (Fig. 7). In state I, this conformation is such that a limited region would adopt an extended structure, thereby giving rise to possible interactions with the peptide binding site of another molecule and thus to self-association. These interactions would mimic interactions between HSC70 and target peptides or proteins and result, in a first step, in the dimerization of the molecule. Self-association does not end with the formation of the dimer, however, and could proceed, leading to polymerization and formation of high molecular weight structures (34, 35, 52). By contrast, state II of the protein, in which the COOH-terminal domain as a whole and/or a limited region adopt an alternative, perhaps more defined, conformation, would be more stable as a monomer than state I and would have a tendency to close on itself as a result of favorable contacts between the NH2- and COOH-terminal domains. That significant interactions should exist between the NH2- and COOH-terminal domain has been proposed by Freeman et al. (9), who showed that the COOH-terminal EEVD sequence plays an important role in the regulation of the NH2-terminal ATPase activity. Furthermore, coiled-coil regions both in the NH2- and COOH-terminal domains, predicted by Lupas et al. (53, 54), could mediate interactions between the two domains.
In conclusion, this scheme reinforces the hypothesis according to which self-association may be a mechanism to regulate the chaperone function of HSC70 (35) and provides a structural basis for this regulation. When HSC70 does not have to "chaperone" another polypeptide chain, its peptide binding site is free but may be protected from becoming bound to unspecific polypeptides by self-association. However, when HSC70 has to assume its chaperone function, specific polypeptide substrates interact with the peptide binding site, thus preventing self-association. In addition, HSC70 monomer-oligomer equilibrium should be submitted to a tight control by the substrates, nucleotides, peptides, and unfolded proteins, as well as regulatory proteins such as the cochaperones that function in concert with HSC70 within the assisted protein folding machinery. ADP as well as cochaperones that promote the ADP-bound state of HSC70, such as DnaJ, would stabilize state I, thus shifting the equilibrium toward self-association, and ATP, peptides, unfolded proteins, or the cochaperones that stabilize the ATP-bound state of HSC70, such as GrpE, would favor state II, thereby promoting monomerization. Although this seems to be the case in vitro (33-35, 52), further investigation is necessary to unravel the biological implications, if any, of HSC70 self-association.
We are very grateful to Bo Fang for help in this work and to Gérard Batelier for assistance in analytical ultracentrifugation experiments.