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Ion mobility–mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility

Nature Chemistry volume 6, pages 208–215 (2014)Cite this article

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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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Figure 1: Flexibility of intact TtATPase and its subcomplexes assessed by IM–MS.
Figure 2: CCS measurements for the TtATPase ICL12 (Vo) and CL12 subcomplexes in different TtATPase preparations.
Figure 3: Nucleotide-associated regulatory mechanism established by IM–MS.
Figure 4: In vacuo MD simulations of the catalytic head (V1′) of TtATPase.

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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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  1. Min Zhou & Argyris Politis

    Present address: Present address: Institute of Chemical Biology and Advanced Materials, Nanjing University of Science and Technology, Nanjing 210094, China (M.Z.); School of Biomedical Sciences, University of Ulster, Londonderry BT47 6SB, UK (A.P.),

  2. Min Zhou and Argyris Politis: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, OX1 3QZ, UK

    Min Zhou, Argyris Politis, Idlir Liko, Kuan-Jung Wu & Carol V. Robinson

  2. The Victor Chang Cardiac Research Institute, Darlinghurst, 2010, NSW, Australia

    Roberta B. Davies, Alastair G. Stewart & Daniela Stock

  3. The University of New South Wales, Sydney, 2052, NSW, Australia

    Roberta B. Davies, Alastair G. Stewart & Daniela Stock

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Contributions

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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Correspondence to Min Zhou or Carol V. Robinson.

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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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