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-assoc iates in solution into dimers, trimers, and probably high order oligom ers, according to a slow temperature- and concentration-dependent equi librium that is shifted toward the monomer upon binding of ATP peptide s or unfolded proteins. To determine the structural basis of HSC70 sel f-association, the oligomerization properties of the isolated amino- a nd carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultrac entrifugation. Whereas the amino-terminal ATPase domain (residues 1-38 4) 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 involv ing monomers, dimers, and trimers. The association equilibrium constan t 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-t erminal domain. Furthermore, oligomerization of the isolated carboxyl- terminal peptide binding domain is, like that of the entire protein, r eversed by peptide binding, indicating that self-association of the pr otein 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 i s in equilibrium between two states differing by the conformation of t heir carboxyl-terminal domain and their self-association properties.