Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate i s not yet understood. Using the glucocorticoid receptor ligand binding doma in as a substrate, we show that dissociation of Hsp90 from bound polypeptid e depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific AT Pase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate rel ease with ATP, but not with the non-hydrolysable nucleotides ATP gammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are p rogressively slower in ATP-dependent substrate release but are still regula ted by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate po orly and show no p23 effect. These results outline an ATP-driven cycle of s ubstrate binding and release for Hsp90 which differs from that of other ATP -driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state t hrough hydrolysis is required for efficient release of substrate polypeptid e. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.