Abstract
Hsp90, a crucial molecular chaperone, significantly influences both normal biology and disease through its regulation of diverse client proteins. Central to its function is its remarkable conformational plasticity, driven by both ATPase activity and client interactions. However, comprehensive insights into Hsp90's dynamic transitions at the molecular level remain elusive. Using solution NMR spectroscopy, here we reveal how ATP binding, hydrolysis, and client engagement drive significant conformational and dynamic shifts in E. coli Hsp90, HtpG, throughout its chaperone cycle. We observe pronounced conformational fluctuations across the entire chaperone molecule, particularly in regions crucial for nucleotide binding and conformational transitions. ATP binding induces an ensemble of slow-exchanging conformations, representing discrete on-path transition states from open to closed forms. Subsequent ATP hydrolysis resolves this structural heterogeneity, shifting HtpG into a distinct, compact conformation. Furthermore, HtpG exhibits stage-specific mobility across the chaperone cycle, potentially enhancing client processing. Client binding acts as an allosteric switch, dynamically priming HtpG for elevated chaperone activity and, therefore, its efficient remodeling. These findings provide atomic-level insights into the functional interplay between Hsp90's conformation, dynamics, nucleotide, and client interactions, culminating in a refined model that elucidates the intricate machinery of Hsp90.