Systematic Mutant Analyses Elucidate General and Client-Specific Aspects of Hsp90 Function

• Functional requirement at each site of N-Hsp90 revealed by systematic mutagenesis
• Conformational constraints depend on amino acid proximity to γ-phosphate of ATP
• The v-src kinase imposes more stringent constraints on hydrolysis compared to GR
• ATPase domain of Hsp90 uses client-specific features to mature different clients

SummaryTo probe the mechanism of the Hsp90 chaperone that is required for the maturation of many signaling proteins in eukaryotes, we analyzed the effects of all individual amino acid changes in the ATPase domain on yeast growth rate. The sensitivity of a position to mutation was strongly influenced by proximity to the phosphates of ATP, indicating that ATPase-driven conformational changes impose stringent physical constraints on Hsp90. To investigate how these constraints may vary for different clients, we performed biochemical analyses on a panel of Hsp90 mutants spanning the full range of observed fitness effects. We observed distinct effects of nine Hsp90 mutations on activation of v-src and glucocorticoid receptor (GR), indicating that different chaperone mechanisms can be utilized for these clients. These results provide a detailed guide for understanding Hsp90 mechanism and highlight the potential for inhibitors of Hsp90 that target a subset of clients.

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