Co-chaperone Regulation of Conformational Switching in the Hsp90 ATPase Cycle

Giuliano Siligardi,Bin Hu,Barry Panaretou,Peter W Piper,Laurence H Pearl,Chrisostomos Prodromou

doi:10.1074/jbc.m410562200

Abstract

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.

Highlights

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer
How the F349A mutation causes a substantial drop in catalytic activity is not certain, but it is likely that Phe349 contributes to the interface between the middle segment and the exposed hydrophobic face of the ATP lid in its closed state
Recent structural studies have shown that p50cdc37 achieves this arrest by binding to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment, fixing the ATP lid in an open conformation and preventing a transactivating interaction of the N-domains [22]

Summary

Introduction

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. The structure of Hsp90 with bound ATP (or AMPNP) has not yet been directly observed, biochemical and mutagenesis studies [6] suggest that, as with GyrB and MutL, the lid segment in Hsp90 closes over bound ATP facilitating N-terminal dimerization [6, 13] and docking with the middle segment of the chaperone [10] to form an active ATPase conformation.

Results

Conclusion