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Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of many client proteins. In order to perform this chaperoning activity Hsp90 hydrolyzes ATP, which requires Mg(II) as cofactor and the hydrolysis is coupled to large global conformational changes. Hsp90 is homo-dimeric with each monomer consisting of three consecutive domains (CTD, MD, NTD). The ATPase site is found in each of the two NTDs, while the CTDs constitute the dimerization site. X-ray crystallography and FRET have provided insights on the conformational cycle of Hsp90 which involves transition from a nucleotide-free ‘open’ to a nucleotide-bound ‘closed’ conformation by dimerization of the NTDs. However, there are still open questions on whether the chaperone shifts global conformation as a consequence of hydrolysis.
Here, we investigate the ATPase site and the concomitant conformational changes at various nucleotide-bound states (pre-hydrolysis, intermediate high energy and post- hydrolysis states) in yeast Hsp90 using EPR techniques. To do so, we substituted the Mg(II) cofactor with paramagnetic Mn(II) and performed hyperfine and pulsed dipolar EPR experiments, to probe short and long range interactions, respectively. Specifically, we tracked ATP hydrolysis by exploring the Mn(II) coordination by the nucleotide phosphates using 31P electron nuclear double resonance (ENDOR) spectroscopy. The interaction of the Mn(II) with protein residues in the different hydrolysis states was investigated by 14/15N ELDOR-detected nuclear magnetic resonance (EDNMR). Last, we measured the distance between the two Mn(II) cofactors in each of the monomers using double electron–electron resonance (DEER/PELDOR) spectroscopy. Here, we measured a well-defined Mn(II)-Mn(II) distance of 4.3 nm in the pre-hydrolysis state, which changes both in width and mean distance in the post-hydrolysis state providing experimental evidence to the existence of two different ‘closed’ conformations for the ATP and ADP bound states. Within our approach one can probe both local and global interactions from a single sample via exploitation of intrinsic sites (here Mg(II)->Mn(II)) that can potentially yield new structural insights previously challenging to observe with FRET and EPR using site-specific spin labeling.