Symmetric stimulation of Hsp90 catalyzed ATP hydrolysis through enhanced active site gate dynamics.

Heat shock protein 90 (Hsp90) is a vital molecular chaperone that is essential for activating a diverse array of regulatory proteins through an ATP-dependent clamping cycle. The Hsp90 clamping cycle is driven by large-amplitude conformational changes within the N-terminal ATPase domain, including the release of an autoinhibitory N-terminal β-strap followed by a less well-characterized ATP gate rearrangement involving N-terminal helix 1. Here, we employed a combination of F NMR spectroscopy, molecular dynamics simulations, and ATPase assays to examine the effects of targeted β-strap and helix 1 mutations. Our findings reveal that targeted disruption of helix 1 packing against the ATPase domain accelerates clamp closure, symmetrically enhancing ATP hydrolysis for both subunits of the Hsp90 dimer, whereas activation by the Aha1 (activator of Hsp90 activity 1) cochaperone is disrupted. Decreasing the energy barrier associated with helix 1 release is a key step in modulating the energy landscape that governs the dynamics of the Hsp90 clamping cycle.