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The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm

Nature volume 495, pages 265–269 (2013)Cite this article

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Abstract

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca2+, initiated by the release of Ca2+ from the sarcoplasmic reticulum and terminated by re-sequestration of Ca2+ into the sarcoplasmic reticulum as the main mechanism of Ca2+ removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN)1,2. Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca2+ enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca2+-ATPase in a previously undescribed E1 state, with exposure of the Ca2+ sites through an open cytoplasmic pathway stabilized by Mg2+. The structure suggests a mechanism for selective Ca2+ loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca2+. These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

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Figure 1: Structure of the SERCA–SLN complex.
Figure 2: Ca 2+ access channel and binding sites.
Figure 3: SERCA–SLN interactions.
Figure 4: Model of the regulation mechanism by SLN.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure have been deposited in the Protein Data Bank under accession number 4H1W.

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Acknowledgements

We thank the staff at beamlines 911-3 at MaxLab and X06SA at the Swiss Light Source. We are grateful to N. Høgholm Jonassen, C. Olesen, B. Nielsen and A.-M. Nielsen for assistance with experimental procedures, and to H. Tidow and P. Gourdon for discussions on autoinhibition. We also thank P. Vangheluwe, H. Young, M. le Maire and E. Carafoli for discussions on PLB and SLN. A.-M.L.W. was supported by postdoctoral fellowships from the Danish Council for Independent Research (Technology and Production Sciences) and the Danish National Advanced Technology Foundation. P.N. is supported by an ERC advanced research grant (BIOMEMOS). Further support was obtained from the Fungalfight and Spotlight projects of the Danish Research Council for Strategic Research.

Author information

Author notes

  1. Anne-Marie L. Winther and Maike Bublitz: These authors contributed equally to this work.

Authors and Affiliations

  1. Pcovery, Thorvaldsensvej 57, DK-1871 Frederiksberg, Denmark,

    Anne-Marie L. Winther, John B. Hansen & Morten J. Buch-Pedersen

  2. Centre for Membrane Pumps in Cells and Disease—PUMPkin, Danish National Research Foundation, Aarhus C, DK-8000, Denmark

    Anne-Marie L. Winther, Maike Bublitz, Jesper L. Karlsen, Jesper V. Møller & Poul Nissen

  3. Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark,

    Anne-Marie L. Winther, Maike Bublitz, Jesper L. Karlsen & Poul Nissen

  4. Department of Biomedicine, Aarhus University, Ole Worms Allé 6, DK-8000 Aarhus, Denmark,

    Jesper V. Møller

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Contributions

A.-M.L.W., J.B.H. and M.J.B.-P. initiated the project. J.V.M. provided sarcoplasmic reticulum membranes and purified SERCA1a, and A.-M.L.W. performed crystallization experiments. Data collection, structure determination and model refinement were performed by M.B. and A.-M.L.W. Videos were prepared by J.L.K. and A.-M.L.W. Model analysis and interpretation was done by M.B., P.N., A.-M.L.W., J.L.K., J.V.M. and M.J.B.-P. The paper was written by M.B., A.-M.L.W., M.J.B.-P. and P.N. with comments from all authors.

Corresponding authors

Correspondence to Poul Nissen or Morten J. Buch-Pedersen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12, which provide detailed close-up views and highlight different aspects of the SERCA–SLN complex structure that are described in detail in the main article and Supplementary Tables 1-3, which list the crystallographic data statistics and the residues in the SERCA–SLN interface with their TM helix assignments and conservation scores. (PDF 2510 kb)

Details of the SERCA–SLN complex structure

This video highlights the novel conformation of the SERCA-SLN complex, particularly at the ion binding site region and at the SERCA-SLN interface. (MOV 27728 kb)

Conformational changes of SERCA along the catalytic cycle

Morphing simulation between structures of the different conformational states of SERCA. (SLN and Mg2+ have been omitted for clarity.) The animation shows the 'sliding door' movement of the kinked M1, which leads to the opening of the Ca2+ entry pathway. Upon closure of the entry pathway and Ca2+-occlusion, contacts between the N- and A-domain are formed by a movement of the N-domain towards the P-domain and a rotation of the A-domain. (MOV 28436 kb)

Model of SLN regulation of SERCA

Morphing simulation of SLN binding to different conformational states of SERCA, assuming that binding is possible in the [Hn]E2 state, but not in [Ca2]E1P. An exchange process of Mg2+ with Ca2+ provides a putative structural basis for a modulatory function of Mg2+ on SERCA activity at high Mg2+ concentrations. (MOV 27353 kb)

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Winther, AM., Bublitz, M., Karlsen, J. et al. The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 495, 265–269 (2013). https://doi.org/10.1038/nature11900

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