25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

EPR probes two 'closed' ATP/ADP conformations and small nucleotide-driven structural changes in yeast Hsp90 chaperone

Not scheduled
Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

Board: 145
Poster Posters


Dr Angeliki Giannoulis (Weizmann Institute of Science)


Hsp90 is a central chaperone performing its activity hydrolyzing ATP with Mg(II) as cofactor. Hsp90 is homo-dimeric and each monomer consists of 3 domains (CTD, MD, NTD). The ATPase site is in the NTDs, while the CTDs are dimerized. In absence of nucleotide Hsp90 is in ‘open’ conformation shifting to an ATP-bound ‘closed’ conformation by dimerization of the NTDs.[1] Yet, there are still open questions on whether the ATP hydrolysis induces global conformational changes and whether the ADP state is ‘open’ or ‘closed’.
We address this question by investigating the local structural changes at the ATPase site and the concomitant conformational changes at various nucleotide-bound states in yeast Hsp90 using EPR techniques. We substituted Mg(II) with Mn(II) and performed hyperfine and pulse dipolar EPR. Specifically, we tracked ATP hydrolysis using 31P ENDOR and investigated Mn(II)-protein interactions by 14/15N EDNMR. We, also, measured the distance between the Mn(II) cofactors using DEER/PELDOR spectroscopy. Using site-directed spin labelling (SDSL) with nitroxide and Gd(III) labels and DEER we monitored inter-monomer rearrangements at different states of the ATPase cycle.
We observed large disorder of Hsp90 as well as spin label flexibility and found that the presence of nucleotide merely dictates the conformational ensemble. This is in contrast to a well-defined Mn(II)-Mn(II) distance in the ATP state, which shortened and broadened in the ADP state, providing experimental evidence to the existence of two different ‘closed’ conformations. The exploitation of the intrinsic metal binding site allowed us probe local and global interactions from a single sample and obtain new structural insights previously challenging to observe with SDSL-DEER/FRET. DEER on mutants revealed small nucleotide-driven structural preferences implying the role of co-chaperones and/or substrates to be the trigger for large conformational changes.

[1] K.A. Verba et al. Science, 2016, 352, 1542; M.M.U. Ali et al. Nature, 2006, 440, 1013

Primary authors

Dr Angeliki Giannoulis (Weizmann Institute of Science) Dr Yoav Barak (Weizmann Institute of Science) Dr Akiva Feintuch (Weizmann Institute of Science) Mr Hisham Mazal (Weizmann Institute of Science) Dr Shira Albeck (Weizmann Institute of Science) Dr Tamar Unger (Weizmann Institute of Science) Prof. Daniella Goldfarb (Weizmann Institute of Science)

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