Structural Basis for Mechanical Coupling in Hsp90: Hinge Flexibility Coordinates ATP Gate Closure and β-Strap Release.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone that relies on coordinated, high-energy, ATP-driven conformational rearrangements to remodel a diverse array of client proteins. A central requirement of the Hsp90 catalytic cycle is the structural coupling between the ATP gate and the N-terminal β-strap. Here, we identify the C-terminal hinge of the ATP gate (G123) as a critical mechanical element mediating this coordination. Using site-specific backbone restriction, 19F nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that hinge plasticity is indispensable for the transition to the closed state and facilitates a primed gate conformation that precedes β-strap release. Restricting hinge flexibility mechanically decouples the β-strap from the ATP gate, trapping Hsp90 in a conformation that precludes chaperone closure. Furthermore, our studies of asymmetric heterodimers demonstrate that hinge rigidity dictates the direction of intersubunit activation or repression across the dimer interface. These findings reveal that the ATP gate hinge functions as a mechanical switch that governs the global conformational state of the Hsp90 complex. These results define a structural basis for Hsp90 activation and highlight how local backbone dynamics drive long-range regulation of the Hsp90 dimer.