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. 2020 Jan 7;117(1):395-404.
doi: 10.1073/pnas.1916030116. Epub 2019 Dec 20.

Two Closed ATP- And ADP-dependent Conformations in Yeast Hsp90 Chaperone Detected by Mn(II) EPR Spectroscopic Techniques

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Two Closed ATP- And ADP-dependent Conformations in Yeast Hsp90 Chaperone Detected by Mn(II) EPR Spectroscopic Techniques

Angeliki Giannoulis et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

Keywords: DEER; EDNMR; ENDOR; Hsp90; chaperone.

Conflict of interest statement

The authors declare no competing interest.

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