“Structural information on Hsp90 by Mn(II)-based EPR”

Organic and Inorganic Chemistry Seminar

Lecturer: Dr. Angeliki Giannoulis, Weizmann Institute of Science

10-10 Jan 2021 @ 09:30

Location: Join Zoom Meeting

Structural information on Hsp90 by Mn(II)-based EPR


  1. Giannoulis, A. Feintuch, Y. Barak, H. Mazal, S. Albeck, T. Unger, F. Yang, X.-C. Su, D. Goldfarb

Heat shock protein of 90 kDa (Hsp90) is an important ATP-dependent molecular chaperone (ATPase) evolutionary conserved in all organisms, preventing misfolding and aggregation of proteins participating in cancer and neurodeneration1. During the ATP hydrolysis cycle Hsp90 undergoes conformational rearrangements, keys aspects of which remain enigmatic while little is known on the interactions of Hsp90 in presence of clients and co-chaperones1.

Using electron paramagnetic resonance (EPR) techniques and substituting the essential Mg(II) cofactor with paramagnetic Mn(II) allowed us employing the double electron-electron resonance (DEER) experiment to probe the distance between the catalytic centers in the pre-hydrolysis, high energy and post-hydrolysis states, which were found different and in the expected range as found by X-ray2-3. Recent DEER experiments in presence of co-chaperone Sba1 revealed a third conformation of Hsp90 in frozen solutions, highlighting the variable landscape the chaperone adopts.

Additionally, we probed for all hydrolysis states the environment of the cofactor with the so-called hyperfine spectroscopy. Specifically, we were able to observe the ATP hydrolysis from the Mn(II)-31P hyperfine coupling signal using the electron nuclear double resonance (ENDOR) experiment and the interaction of Mn(II) with neighboring atoms (1H, 31P, 14N) using eldor detected NMR (EDNMR).

The substitution of Mg(II) with Mn(II) was critical in identifying three structurally proximate, however different conformations of Hsp90, and concomitantly observe the local changes at the catalysis site. The methodology can be applied to all molecular machines using Mg(II)/ATP as fuel.



  1. Taipale, M.; Jarosz, D. F.; Lindquist, S., HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell. Bio. 2010, 11 (7), 515-528.
  2. Ali, M. M. U.; Roe, S. M.; Vaughan, C. K.; Meyer, P.; Panaretou, B.; Piper, P. W.; Prodromou, C.; Pearl, L. H., Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 2006, 440 (7087), 1013-1017.
  3. Verba, K. A.; Wang, R. Y.; Arakawa, A.; Liu, Y.; Shirouzu, M.; Yokoyama, S.; Agard, D. A., Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 2016, 352 (6293), 1542-1547.