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The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis

Abstract

The molecular chaperone heat-shock protein 90 (Hsp90) is one of the most abundant proteins in unstressed eukaryotic cells. Its function is dependent on an exceptionally slow ATPase reaction that involves large conformational changes. To observe these conformational changes and to understand their interplay with the ATPase function, we developed a single-molecule assay that allows examination of yeast Hsp90 dimers in real time under various nucleotide conditions. We detected conformational fluctuations between open and closed states on timescales much faster than the rate of ATP hydrolysis. The compiled distributions of dwell times allow us to assign all rate constants to a minimal kinetic model for the conformational changes of Hsp90 and to delineate the influence of ATP hydrolysis. Unexpectedly, in this model ATP lowers two energy barriers almost symmetrically, such that little directionality is introduced. Instead, stochastic, thermal fluctuations of Hsp90 are the dominating processes.

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Figure 1: Experimental setup.
Figure 2: Single-molecule FRET time traces with ATP.
Figure 3: Dwell-time distributions for various nucleotide conditions.
Figure 4: smFRET trace with AMP-PNP and the Δ8 mutant.
Figure 5: Minimal kinetic model.
Figure 6: Rate constants, uncertainties and energy landscape.

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Acknowledgements

We thank E. Frey, R. Metzler, K. Richter and M. Rief for helpful discussions and critical reading of the manuscript and Nano Initiative Munich for financial support.

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Contributions

M.M. performed experiments and kinetic Monte Carlo calculations; M.H., M.M. and C.R. designed constructs and purified the proteins. M.M. and C.R. labeled and characterized the proteins; J.B. and T.H. planned and supervised the study; T.H. and M.M. wrote the manuscript; all authors discussed the results and commented on the manuscript.

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Correspondence to Thorsten Hugel.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 370 kb)

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Mickler, M., Hessling, M., Ratzke, C. et al. The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis. Nat Struct Mol Biol 16, 281–286 (2009). https://doi.org/10.1038/nsmb.1557

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