Mechanistic insight to the Hsp90 chaperone by mutational analysis of conserved interaction sites

Monday, September 22, 2014 — Poster Session II
4:00 p.m. – 6:00 p.m.	FAES Academic Center	NCI	MOLBIO-9

Authors

• A.N. Kravats
• O. Genest
• J.R. Hoskins
• S. Wickner

Abstract

Heat shock protein 90(Hsp90) is a molecular chaperone that protects client proteins from misfolding and aggregation. It assembles into a homodimer, with each protomer comprised of an N-terminal, middle(M), and C-terminal domain. Hsp90 undergoes ATPase activity, resulting in large-scale conformational rearrangements used to remodel client proteins. Cochaperones also interact with different Hsp90 domains to either stimulate or inhibit ATPase activity. Though structure and function of Hsp90 are widely conserved, the interaction mechanism between Hsp90 and a diverse set of clients is unclear. We used a bacterial two-hybrid assay to identify residues of E.coli Hsp90(Hsp90Ec) that were required for interaction with DnaK, an Hsp70 homolog. These residues reside on the M-domain surface and the mutant proteins were defective in luciferase reactivation. To determine whether this site on Hsp90Ec is functionally conserved, we made substitutions of homologous residues in yeast Hsp90(Hsp82). The Hsp82 mutant proteins were purified and tested in protein reactivation assays and in ATPase assays with and without co-chaperones. The results indicate a lower rate of client reactivation by the Hsp82 mutants. Also, Hsp82 mutants exhibit defective ATPase activity in the presence of cochaperones Aha1 and Sti1, suggesting this region is involved in an important protein-protein interaction or conformational change.

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