Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field F NMR Spectroscopy.

Protein turnover in cells is regulated by the ATP dependent activity of the Hsp90 chaperone. In concert with accessory proteins, ATP hydrolysis drives the obligate Hsp90 dimer through a cycle between open and closed states that is critical for assisting the folding and stability of hundreds of proteins. Cycling is initiated by ATP binding to the ATPase domain, with the chaperone and the active site gates in the dimer in open states. The chaperone then adopts a short-lived, ATP bound closed state with a closed active site gate. The structural and dynamic changes induced in the ATPase domain and active site gate upon nucleotide binding, and their impact on dimer closing are not well understood. We site-specifically F-labeled the ATPase domain at the active site gate to enable benchtop and high field F NMR spectroscopic studies. Combined with MD simulations, this allowed accurate characterization of pico- to nanosecond time scale motions of the active site gate, as well as slower micro- to millisecond time scale processes resulting from nucleotide binding. ATP binding induces increased flexibility at one of the hinges of the active site gate, a necessary prelude to release of the second hinge and eventual gate closure in the intact chaperone.