Featured Article - December 2014
Short description: An asymmetric Hsp90 structure supports a new mechanism for coupling ATP hydrolysis with substrate turnover.
Hsp90 chaperones assist in protein folding and maintaining cellular proteostasis, and their activity is increasingly linked to diseases such as cancer. Hsp90s contain three domains: the N-terminal domain, where ATP binding and hydrolysis occurs; the 'middle' domain, which assists in ATP hydrolysis; and the C-terminal domain, responsible for homodimerization. Hsp90s undergo large-scale conformational motions during their catalytic cycle, with growing evidence that these movements are asymmetric. However, the basis for this asymmetry, as well as the mechanism by which ATP hydrolysis drives the binding and release of substrate proteins, is not well understood.
Agard and colleagues (PSI Protein Homeostasis) explore these questions in a structural and biochemical analysis of TRAP1, the Hsp90 homolog in the mitochondria. The authors first solved 2.3-Å resolution structures of TRAP1 with three ATP analogues (PDB 4IPE, 4IYN and 4J0B). In contrast to the previously solved closed structure of an AMPPNP and co-chaperone-bound Hsp90 from yeast, the authors observed asymmetry between the monomers. In particular, the middle domain was rotated relative to the N- and C-terminal domains in one monomer as compared to the other. This unusual twisting motion was constrained by strong contacts across both the C-terminal domain, as expected, and the N-terminal domain.
Additional spectroscopic and biochemical studies supported the structural findings: small-angle X-ray scattering measurements matched an asymmetric structure better than the symmetric structure for both TRAP1 and a previously studied bacterial Hsp90 homolog. Dipolar electron-electron resonance analysis of paramagnetically labeled constructs similarly supported the existence of two states. The mutation of residues involved in stabilizing both the twist and differential exposure of known substrate binding residues inhibited ATPase activity and impaired binding of a model substrate (Δ131Δ), supporting the functional relevance of the motions.
Notably, a crystal structure of a construct lacking the C-terminal domain (PDB 4IVG) regained symmetry, presumably because the restraining interfacial interactions were lost. Altogether, the data lead to a model where different ATP hydrolysis steps could occur via two distinct closed conformations: the first hydrolysis causes a change from an asymmetric to another (possibly symmetric) closed state, during which substrate reorganization could take place, while the second hydrolysis event leads to substrate release.
L.A. Lavery et al. Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism.
Mol. Cell 53, 330-343 (2014). doi:10.1016/j.molcel.2013.12.023