Structural Asymmetry in the Closed State of Mitochondrial Hsp90 (TRAP1) Supports a Two-Step ATP Hydrolysis Mechanism

While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface. This interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity and substrate binding and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release. [Display omitted] •Crystal structure of the TRAP1 homodimer reveals an asymmetric closed state•SAXS and DEER validate asymmetry in solution and conservation across Hsp90 homologs•MD:CTD interfaces are functional for chaperone activity and stabilize asymmetry•Changes in asymmetry result in rearrangement of client binding residues Hsp90 is critical to many signaling pathways and functions through affecting the state of its “clients.” Lavery et al. present crystal structures of the mitochondrial homolog in a catalytically active conformation, revealing an asymmetric state. They show that this state is functional for chaperone activity and propose a model that utilizes structural asymmetry for client remodeling.