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Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

Nature Chemical Biology volume 12, pages 628–635 (2016)Cite this article

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Abstract

The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

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Figure 1: Observation of conformational motions in Hsp90 by PET fluorescence quenching.
Figure 2: Modulation of conformational motions by mutagenesis.
Figure 3: Influence of Aha1 on kinetics of local motions.
Figure 4: Equilibrium dynamics of lid and N-terminal β-strand probed by PET-FCS.
Figure 5: Possible origins of multi-exponential kinetics of Hsp90 conformational change.
Figure 6: Integration of results into the chaperone catalytic cycle.

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Acknowledgements

We thank the Deutsche Forschungsgemeinschaft (grant NE 1201/3-1 to H.N.) and the Wellcome Trust (Senior Investigator Award 095605/Z11/Z to L.H.P.). A.S. was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences (University of Würzburg).

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Authors and Affiliations

  1. Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.,

    Andrea Schulze, Gerti Beliu, Dominic A Helmerich, Jonathan Schubert & Hannes Neuweiler

  2. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK.,

    Laurence H Pearl & Chrisostomos Prodromou

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  1. Andrea Schulze
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Contributions

A.S. designed experiments, synthesized modified protein material, performed rapid-mixing fluorescence experiments, performed ATPase assays, analyzed data, interpreted results and wrote the paper. G.B. synthesized modified protein material, performed PET-FCS experiments, analyzed data and interpreted results. D.A.H. synthesized modified protein material, performed PET-FCS experiments and analyzed data. J.S. synthesized modified protein material, performed PET-FCS experiments and analyzed data. L.H.P. interpreted results and wrote the paper. C.P. designed experiments, interpreted results and wrote the paper. H.N. conceptually designed the research, designed experiments, analyzed data, interpreted results and wrote the paper.

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Correspondence to Hannes Neuweiler.

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Schulze, A., Beliu, G., Helmerich, D. et al. Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism. Nat Chem Biol 12, 628–635 (2016). https://doi.org/10.1038/nchembio.2111

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