Nucleotide-Dependent Interaction of Saccharomyces cerevisiae Hsp90 with the Cochaperone Proteins Sti1, Cpr6, and Sba1

Abstract

The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.

We thank Elizabeth Craig, Susan Lindquist, Avrom Caplan, and Brian Freeman for reagents. We also thank David Toft and Elizabeth Craig for helpful advice pertaining to the manuscript.

This publication was made possible by grant no. P20 RR15587 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). This project was also funded in part by an American Cancer Society seed grant through Washington State University, IRG-77-003-26.

The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.