Asymmetric Dynamics Drive Catalytic Activation of the Hsp90 Chaperone

The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended