The conformationally coupled mechanism by which ATP is utilized by yeast Hsp90 is now well characterized. In contrast, ATP utilization by human Hsp90s is less well studied, and appears to operate differently. To resolve these conflicting models, we have conducted a side-by-side biochemical analysis in a series of mutant yeast and human Hsp90s that have been both mechanistically and structurally characterized with regard to the crystal structure of the yeast Hsp90 protein. We show that each monomer of the human Hsp90 dimer is mutually dependent on the other for ATPase activity. Fluorescence studies confirmed that the N-terminal domains of Hsp90beta come into close association with each other. Mutations that directly affect the conformational dynamics of the ATP-lid segment had marked effects, with T31I (yeast T22I) and A116N (yeast A107N) stimulating, and T110I (yeast T101I) inhibiting, human and yeast ATPase activity to similar extents, showing that ATP-dependent lid closure is a key rate-determining step in both systems. Mutation of residues implicated in N-terminal dimerization of yeast Hsp90 (L15R and L18R in yeast, L24R and L27R in humans) significantly reduced the ATPase activity of yeast and human Hsp90s, showing that ATP-dependent association of the N-terminal domains in the Hsp90 dimer is also essential in both systems. Furthermore, cross-linking studies of the hyper-active yeast A107N and human A116N ATP-lid mutants showed enhanced dimerization, suggesting that N-terminal association is a direct consequence of ATP binding and lid closure in both systems.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|