IN-VIVO FUNCTION OF HSP90 IS DEPENDENT ON ATP BINDING AND ATP HYDROLYSIS

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regula tory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In parti cular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 est ablished the presence of a conserved nucleotide binding site that is i dentical with the binding site of geldanamycin, a specific inhibitor o f Hsp90. The functional significance of nucleotide binding by Hsp90 ha s remained unclear. Here we present evidence for a slow but dearly det ectable ATPase activity in purified Hsp90. Based on a new crystal stru cture of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Es cherichia coli DNA gyrase, the residues of Hsp90 critical in ATP bindi ng (D93) and ATP hydrolysis (E47) were identified. The corresponding m utations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results sh ow that both ATP binding and hydrolysis are required for Hsp82 functio n in vivo. The mutant Hsp90 proteins tested are defective in the bindi ng and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypepti de from Hsp90, Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intric ate allosteric communication between the domains of the Hsp90 dimer. O ur results establish Hsp90 as an ATP-dependent chaperone.