Article

Dynamics of P-type ATPase transport revealed by single-molecule FRET

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Abstract

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

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Acknowledgements

We thank J. V. Møller and the members of the Blanchard and Nissen laboratories for helpful discussions, A. M. Nielsen, T. Klymchuk and L. T. Pedersen for technical assistance, and J. Juul and J. Karlsen for key support on molecular animations. Support for this work was provided by grants from the NIH to S.C.B. (grant number 1R01GM098859), by an EMBO Short-Term Fellowship to M.D., and by Lundbeck Foundation and AIAS COFUND fellowships to M.K. We also thank the Molecular Machines COST Action (CM1306) for helpful support.

Author information

Author notes

    • Mateusz Dyla
    •  & Daniel S. Terry

    These authors contributed equally to this work.

Affiliations

  1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Danish National Research Foundation & Danish Research Institute of Translational Neuroscience – DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, DK-8000 Aarhus C, Denmark

    • Mateusz Dyla
    • , Magnus Kjaergaard
    • , Jacob Lauwring Andersen
    •  & Poul Nissen
  2. Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark

    • Mateusz Dyla
    • , Magnus Kjaergaard
    • , Jacob Lauwring Andersen
    • , Charlotte Rohde Knudsen
    •  & Poul Nissen
  3. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark

    • Mateusz Dyla
    • , Magnus Kjaergaard
    •  & Poul Nissen
  4. Department of Physiology and Biophysics, Weill Cornell Medicine, Cornell University, New York, New York 10021, USA

    • Daniel S. Terry
    • , Roger B. Altman
    •  & Scott C. Blanchard
  5. Aarhus Institute of Advanced Studies (AIAS), Aarhus University, DK-8000 Aarhus C, Denmark

    • Magnus Kjaergaard
  6. Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK

    • Thomas L.-M. Sørensen
  7. Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark

    • Jens Peter Andersen

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Contributions

P.N. conceived the study. M.D., with assistance from M.K., J.L.A. and C.R.K., performed site-directed mutagenesis, protein expression, purification, labelling and ATPase activity experiments. M.D. and T.L.-M.S., with guidance from J.P.A., performed and analysed the phosphorylation experiments. D.S.T., with help from S.C.B. and R.B.A., conceived and performed the single-molecule FRET experiments and analysed the data. P.N. and S.C.B. supervised the project. All authors wrote the manuscript.

Competing interests

S.C.B. and R.B.A. have an equity interest in Lumidyne Technologies.

Corresponding authors

Correspondence to Poul Nissen or Scott C. Blanchard.

Reviewer Information Nature thanks G. Bultnyck, S. Karlish and A. van Oijen for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Tables

    This file contains Supplementary Table 1 (distance changes expected from crystal structure models) and Supplementary Table 2 (dose-response fit parameters).

Videos

  1. 1.

    Conformational changes of SERCA time-scaled to visualize LMCA1 dynamics.

    The observed dynamics of the LMCA1 cycle have been applied and scaled to a morphed series of conformational changes in rabbit SERCA1a based on the following PDB accession numbers (functional states): 4H1W (E1), 1T5S ((Ca)E1-ATP), 1T5T ([Ca]E1P-ADP), 3B9B (E2P), 3B9R ([H]E2P dephosphorylation), 2C88 ([H]E2). The relative transition rates applied to the morph were based on the data summarized in Fig. 4.