Hsp90, a dimeric ATP-dependent molecular chaperone, is required for the folding and activation of numerous essential substrate "client" proteins including nuclear receptors, cell cycle kinases, and telomerase. Fundamental to its mechanism is an ensemble of dramatically different conformational states that result from nucleotide binding and hydrolysis and distinct sets of interdomain interactions. Previous structural and biochemical work identified a conserved arginine residue (R380 in yeast) in the Hsp90 middle domain (MD) that is required for wild type hydrolysis activity in yeast, and hence proposed to be a catalytic residue. As part of our investigations on the origins of species-specific differences in Hsp90 conformational dynamics we probed the role of this MD arginine in bacterial, yeast, and human Hsp90s using a combination of structural and functional approaches. While the R380A mutation compromised ATPase activity in all three homologs, the impact on ATPase activity was both variable and much more modest (2-7 fold) than the mutation of an active site glutamate (40 fold) known to be required for hydrolysis. Single particle electron microscopy and small-angle X-ray scattering revealed that, for all Hsp90s, mutation of this arginine abrogated the ability to form the closed "ATP" conformational state in response to AMPPNP binding. Taken together with previous mutagenesis data exploring intra- and intermonomer interactions, these new data suggest that R380 does not directly participate in the hydrolysis reaction as a catalytic residue, but instead acts as an ATP-sensor to stabilize an NTD-MD conformation required for efficient ATP hydrolysis.
|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|