Abstract
Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.
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Acknowledgements
This work was funded by the Wellcome Trust (M.Z. and C.V.R.) and by a PROSPECTS (HEALTHF4-2008-201648) grant within the Research Framework of the European Union (A.P. and C.V.R.), together with Engineering and Physical Sciences Research Council funding for research scientists with caring responsibilities (M.Z.), funding from the Royal Society and an European Research Council investigator award Integral Membrane Proteins Resolution of Stoichiometry and Structure (IMPRESS) (C.V.R.). D.S., R.B.D. and A.G.S. are funded by Australian National Health and Medical Research Council grants 1004620, 1022143 and 1047004, respectively.
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C.V.R. and D.S designed the research, M.Z. conducted all IM–MS experiments and analysed the data, R.B.D. purified TtATPases, A.P. and A.G.S. performed the modelling, A.P. conducted MD simulations and all CCS calculations, I.L. carried out the MS analysis of dAb:TtATPase binding, K.J.W. conducted the Western blot experiments, and M.Z. and C.V.R. and A.P. wrote the paper.
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Zhou, M., Politis, A., Davies, R. et al. Ion mobility–mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility. Nature Chem 6, 208–215 (2014). https://doi.org/10.1038/nchem.1868
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DOI: https://doi.org/10.1038/nchem.1868
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