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.