Mammalian cell sensitivity to hyperthermia in various cell lines: a new universal and predictive description.

Introduction: The Cumulative Equivalent Minute at 43 °C (CEM43) thermal dose model has been empirically derived more than 30 years ago and still serves as a benchmark for hyperthermia protocols despite the advent of regulatory network models. However, CEM43 suffers from several limitations regarding its inability to predict the effect of complex time varying profiles (thermotolerance, step-down heating), to predict synergistic effects with drug treatments or to explain the specificity of a cell line in thermal resistance.Objective: Define a new generic predictive tool for thermal injury based on regulatory network models. Identify the biological parameters that account for the thermal resistance.Materials: Comparative study of cell survival upon hyperthermia collected from literature (17 sets in 11 publications that cover 14 different cell lines from 8 different tissues).Results: A dynamical model describes accurately cell survival according to the amplitude and duration of exposure but also molecular chaperone expression level. In the case of square shape hyperthermia, approximated analytical expression of the cell survival is derived from the dynamical model and compared to CEM43 description. The molecular chaperone expression level defines the thermal resistance of a given cell line and can be estimated from a single experimental result through an easy-to-use graphical tool.Conclusion: The tools offered here can be useful for designing treatments combining hyperthermia and chemotherapy targeting molecular chaperones, but also for designing personalized hyperthermic treatment by prior biochemical screening of molecular chaperones. These tools could advantageously replace the description of CEM43.

Dynamical thermal dose models and dose time-profile effects.

Introduction: Models of dose-effect relationships seek systematic and predictive descriptions of how cell survival depends on the level and duration of the stressor. The CEM43 thermal dose model has been empirically derived more than thirty years ago and still serves as a benchmark for hyperthermia protocols despitethe advent of regulatory network models. Objective: In this paper, we propose and realize a simple experimental test to assess whether mechanistic models can prove more reliable indicators for some protocols. We define two time-asymmetric hyperthermia profiles, faster rise than decay or slower rise than decay, for which the CEM43 model predicts the same survival while a regulatory network model predicts significant differences. Materials: Experimental data (both control 37°C and hyperthermia assays) were collected from duplicate HeLa cell cultures. Cells were imaged before and 24, 48 and 72 h after the hyperthermia assay double-stained with fluorescein-5-isothiocyanate (FITC)-labeled annexin V and propidium iodide for detecting cell death. Results: Survival experiments of HeLa cells show that a fast temperature rise followed by a slow decay can be twice more lethal than the opposite, consistently with the prediction of the network model. Conclusions: Using a model reduction approach, we obtained a simple nonlinear dynamic equation that identifies the limited repair capacity as the main factor underlying the dose-asymmetry effect and that could be useful for refining thermal doses for dynamic protocols.

Induction of Hsp 70 in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shock and by Vitamin E.

This paper analysed the toxicity mechanisms of several mycotoxins using Hsp 70 expression, cytoprotection of Vero cells by sub-lethal heat shock (sub-LHS) and Vitamin E. Our aim was (i) to determine whether Citrinin (CTN), Zearalenone (ZEN) and T2 toxin (T2) could induce the expression of Hsp 70, (ii) to check whether or not elevated levels of Hsp and Vitamin E pre-treatment could provide cytoprotection from these mycotoxins, and finally (iii) to emphasize the eventual involvement of oxidative stress on mycotoxin's toxicity. Our study demonstrated that the three examined mycotoxins induced Hsp 70 expression in a dose-dependent manner. A cytoprotective effect of Hsp 70 was obtained when Vero cells were exposed to sub-lethal heat shock followed by a 12h recovery prior to mycotoxins treatment and evidenced by a reduction of their cytolethality. This cytoprotection suggested that Hsp 70 might constitute an important cellular defence mechanism. A cytoprotective action was also obtained although at lesser extent, when cells were pre-treated with an antioxidant agent, the Vitamin E before mycotoxins treatment. This Vitamin E cytoprotection evoked the involvement of oxidative stress in mycotoxins induced toxicity, which was further, confirmed by the reduction of Hsp 70 expression when cells were pre-treated with Vitamin E prior to mycotoxins. Our data clearly shows that oxidative stress is certainly involved in the toxicity of the three studied mycotoxins, Citrinin, Zearalenone and T2 toxin and may therefore constitutes a relevant part in their toxicities; however, at variable extent from one mycotoxin to another.

Involvement of the interdomain hydrophobic linker and the C-terminal helices in self-association of the molecular chaperone HSC70.

HSP70 from bacteria to man are known to self-associate to form multiple species suggesting that self-association is related to function. In order to determine the structural basis of HSP70 oligomerization, deletion mutants in the C-terminal domain of HSC70, a constitutive member of the HSP70 family, have been constructed and analyzed for their self-association properties by gel electrophoresis, size-exclusion chromatography and analytical ultracentrifugation. The results of this investigation indicate that, whereas deletion of the GGMP rich C-terminal extremity of HSC70, containing EEVD motif stabilizes the oligomeric species, deletions of either the aD-aE C-terminal helices or the inter-domain hydrophobic linker contribute to the stabilization of the monomeric form. Thus, two non-contiguous regions, located at both ends of the C-terminal domain of the protein, appear to form the contact interface in the oligomers and may interact in a dynamic fashion leading to the formation of several coexisting species.

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. II. Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions.

The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al. has been purified to homogeneity. In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the lambda DNA. This modification has very informative consequences on the allosteric properties of ATCase. pAR5-ATCase lacks the homotropic co-operative interactions between the catalytic sites for aspartate binding and is "frozen" in the R state. In addition, this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP, in spite of the fact that this nucleotide binds normally to the regulatory sites. Conversely, pAR5-ATCase is fully sensitive to the activator ATP. However, this activation is limited to the extent of the previously described "primary effect" as expected from an ATCase form "frozen" in the R state. These results emphasize the importance of the three-dimensional structure of the C-terminal region of the regulatory chains for both homotropic and heterotropic interactions. In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area.

The activation of Escherichia coli aspartate transcarbamylase by ATP. Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains.

The regulatory chain of E. coli aspartate transcarbamylase (E.C. 2.1.3.2) is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.

Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains.

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation.

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

Intramolecular transmission of the ATP regulatory signal in Escherichia coli aspartate transcarbamylase: specific involvement of a clustered set of amino acid interactions at an interface between regulatory and catalytic subunits.

Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP. Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites. The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP. These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain. Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding. These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model. This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.

Cytotoxicity and Hsp 70 induction in Hep G2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock.

Zearalenone (ZEN) is a mycotoxin with several adverse effects in laboratory and domestic animals. The mechanism of ZEN toxicity that involves mainly binding to oestrogen receptors and inhibition of macromolecules synthesis is not fully understood. Using human hepatocytes Hep G2 cells as a model, the aim of this work was (i) to investigate the ability of ZEN to induce heat shock proteins Hsp 70 and (ii) to find out the mechanisms of ZEN cytotoxicity by examining cell proliferation and protein synthesis. Our study demonstrated that ZEN induces Hsp 70 expression in a time and dose-dependant manner; this induction occurs at non-cytotoxic concentrations, it could be therefore considered as a biomarker of toxicity. A cytoprotective effect of Hsp 70 was elicited when Hep G2 cells were exposed to Sub-Lethal heat shock prior to ZEN treatment and evidenced by a reduced ZEN cytolethality. This cytoprotection suggests that Hsp 70 may constitute an important cellular defence mechanism. Finally, our data show that ZEN is cytotoxic in Hep G2 cells by inhibiting cell proliferation and total protein synthesis and pointed out oxidative damage as possible pathway involved in ZEN toxicity; however, other investigations are needed to further confirm Zen induced oxidative stress.

Engineering aspartate transcarbamylase.

Aspartate transcarbamylase from Escherichia coli is one of the most extensively studied regulatory enzymes as a model of cooperativity and allostery. Numerous methods are used to engineer variants of this molecule: random and site-directed mutagenesis, dissociation and reassociation of the catalytic and regulatory subunits and chains, construction of hybrids made from normal and modified subunits or chains, interspecific hybrids and construction of chimeric enzymes. These methods provide detailed information on the regions, domains, interfaces and aminoacid residues which are involved in the mechanism of co-operativity between the catalytic sites, and of regulation by the antagonistic effectors CTP and ATP. These effectors induce the transmission of intramolecular signals whose pathways begin to be delineated.

A possible model for the concerted allosteric transition in Escherichia coli aspartate transcarbamylase as deduced from site-directed mutagenesis studies.

Aspartate transcarbamylase is stabilized in a low-affinity-low-activity state exhibiting no cooperativity by selective perturbation of the Glu-50-Arg-167 and Glu-50-Arg-234 interdomain salt bridges. Similarly, a high-affinity-high-activity state of the enzyme, retaining a significant amount of cooperativity, is obtained by perturbation of the interaction between Tyr-240 and Asp-271. In this work, we show that the rupture of the link between Tyr-240 and Asp-271 in the enzyme already lacking the interdomain salt bridges regenerates the homotropic cooperative interactions between the catalytic sites and substantially increases the activity and affinity of the enzyme for aspartate. These results suggest a possible relationship between these two sets of interactions for the establishment of the cooperative behavior of the enzyme. Another mutation, Glu-239 to Gln, introduced to perturb the Glu-239-Lys-164 and Glu-239-Tyr-165 interactions between the two catalytic subunits, is sufficient to "lock" the enzyme in the R state. These observations emphasize the importance of the interactions at the interface between the catalytic trimers in maintaining the T state of the enzyme and shed light on the role played by this pathway in the communication of homotropic cooperativity between the different sites. A model including all these findings, as well as the interactions stabilizing the T state or the R state in the presence of the natural substrates, is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase.

The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263). In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine. This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation. The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme. Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity. The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme. Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate. Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme. These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.

Site-directed mutagenesis of a residue located in the regulatory site of Escherichia coli aspartate transcarbamoylase. Involvement of lysine 94 in effector binding and the allosteric mechanism.

Lysine 94 in the regulatory chain of aspartate transcarbamoylase has been changed to a glutamine residue by site-directed mutagenesis. The resulting enzyme is almost insensitive to the activator ATP and shows a substantially reduced response to the feedback inhibitor CTP. Competition experiments indicate that ATP is still able to bind at low concentrations to the regulatory site of the mutant enzyme, even though no stimulation could be detected. When the nucleosides adenosine or cytidine were used, the saturation curves of the mutant and the wild-type enzyme became indistinguishable. Together these results indicate that lysine 94 is strongly involved in the binding of ATP and CTP by interacting specifically with the triphosphate moiety of these nucleotide effectors. Furthermore, unlike the wild-type enzyme, the inhibitory and stimulatory effects in the mutant enzyme are insensitive to changes in aspartate concentrations, implying that the lysine 94 side chain is also involved in the allosteric mechanism of the enzyme.

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli.

The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.

The COOH-terminal peptide binding domain is essential for self-association of the molecular chaperone HSC70.

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 10(5) M-1, 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.

Effect of nucleotides, peptides, and unfolded proteins on the self-association of the molecular chaperone HSC70.

In a previous study, we showed that the molecular chaperone HSC70 self-associates in solution in a reversible and likely unlimited fashion. Here, we examine the influence of nucleotides, nucleotide analogs, peptides, and unfolded proteins on the self-association properties of this protein. Whereas in the presence of ADP, HSC70 exists as a slow, concentration- and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, the protein is essentially monomeric, indicating that ATP shifts this equilibrium toward the monomer by stabilizing the monomer. Dissociation of oligomers into monomers is also obtained with the slowly hydrolyzable ATP analogs, adenosine 5'-O-(thiotriphosphate) and 5'-adenylyl-beta,gamma-imidodiphosphate, or the complex between ADP and the phosphate analog, BeF3, indicating that binding but not hydrolysis of ATP is necessary and sufficient for the stabilization of HSC70 monomer. Furthermore, binding of short peptides or permanently unfolded proteins to the peptide binding site of HSC70 promotes the dissociation of oligomers into monomers, suggesting that protein substrates are able to compete with HSC70 for the same binding site. Because the release of peptides or unfolded proteins from HSC70 has also been shown to require ATP binding, these results indicate that dissociation of oligomers is controlled by a mechanism similar to that of release of protein substrates and suggest that binding of HSC70 to itself occurs via the peptide binding site and mimics binding of HSC70 to protein substrates.

Unlike the quaternary structure transition, the tertiary structure change of the 240s loop in allosteric aspartate transcarbamylase requires active site saturation by substrate for completion.

The quaternary structural change associated with the homotropic cooperative interactions in Escherichia coli aspartate transcarbamylase (ATCase) is accompanied by various tertiary structural modifications; the most notable one involves the 240s loop formed by residues 230--245 of the catalytic chain. In order to monitor local conformational changes in this region by fluorescence spectroscopy, Tyr-240 has been replaced by a Trp residue, in a mutant enzyme, in which both naturally occurring Trp residues in positions 209 and 284 of the catalytic chains had previously been substituted by Phe residues. This F209F284W240-ATCase still displays homotropic cooperativity for aspartate and undergoes the same T to R quaternary structure change as does the wild-type enzyme. Upon binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate, the fluorescence emission spectrum of this mutant shows a red shift directly proportional to the fraction of catalytic sites occupied by this compound, a maximum value of 4 nm being attained when all six active sites are ligated. An identical shift is observed with the catalytic subunits of this modified enzyme, when all three active sites are occupied. In contrast, the quaternary structural change of the F209F284W240-ATCase, monitored by small-angle X-ray scattering, is complete when only four out of six catalytic sites are occupied. Thus, the 240s loop adopts its final conformation only when the neighboring active site is bound.

Self-association of the molecular chaperone HSC70.

The self-association properties of the molecular chaperone HSC70 have been analyzed by a wide range of biochemical and biophysical techniques. Nondenaturing gel electrophoresis and cross-linking studies show the presence of multiple species going from monomer to at least trimer. Size-exclusion chromatography gives two overlapping peaks, a major one corresponding to species having the molecular mass of monomer (70 kDa) and a minor broad one corresponding to species with a molecular mass range of 150-300 kDa. Progressive dilution of the protein leads to an increase in the size of the monomer peak at the expense of that of the oligomeric peak, thus indicating a concentration-dependent chemical equilibrium. Sedimentation velocity reveals the presence of three species, whose proportions were dependent on concentration, but whose sedimentation coefficients, s20,w, of 4.3, 6.6, and 8.5 S did not vary with concentration, indicative of a slowly equilibrating system. Sedimentation equilibrium studies confirmed these results and showed a dissociation into monomers at low concentrations and an association into dimers and trimers at high concentrations. The multiple sedimentation equilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equilibrium constants by a monomer-dimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium were respectively K1-2 = 1.1 x 10(5) M-1 and K2-3 = 0.9 x 10(5) M-1. Interestingly, an isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2 x 10(5) M-1. (ABSTRACT TRUNCATED AT 250 WORDS)

Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70.

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.