Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The structural basis of calcium transport by the calcium pump

Abstract

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overall comparison of SERCA1a structures representing key states of the reaction cycle.
Figure 2: The Ca2E1P state obtained with AMPPNP.
Figure 3: The E2P state obtained with beryllium fluoride.
Figure 4: The luminal exit pathway of sarcoplasmic reticulum Ca 2+ -ATPase.
Figure 5: Changes at the phosphorylation site of SERCA1a in the functional cycle.
Figure 6: Schematic representation of the reaction cycle.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Rolfe, D. F. & Brown, G. C. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol. Rev. 77, 731–758 (1997)

    CAS  Article  PubMed  Google Scholar 

  2. Carafoli, E. Calcium signaling: a tale for all seasons. Proc. Natl Acad. Sci. USA 99, 1115–1122 (2002)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  3. Ebashi, S. & Lipmann, F. Adenosine triphosphate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. J. Cell Biol. 14, 389–400 (1962)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Hasselbach, W. Quantitative aspects of the calcium concept of excitation contraction coupling—a critical evaluation. Basic Res. Cardiol. 75, 2–12 (1980)

    CAS  Article  PubMed  Google Scholar 

  5. De Meis, L. The sarcoplasmic reticulum: Transport and Energy Transduction (ed. Bittar, E. E.) (Wiley & Sons, New York, 1981)

    Google Scholar 

  6. Levy, D., Seigneuret, M., Bluzat, A. & Rigaud, J. L. Evidence for proton countertransport by the sarcoplasmic reticulum Ca2+-ATPase during calcium transport in reconstituted proteoliposomes with low ionic permeability. J. Biol. Chem. 265, 19524–19534 (1990)

    CAS  PubMed  Google Scholar 

  7. Cornelius, F. & Moller, J. V. Electrogenic pump current of sarcoplasmic reticulum Ca2+-ATPase reconstituted at high lipid/protein ratio. FEBS Lett. 284, 46–50 (1991)

    CAS  Article  PubMed  Google Scholar 

  8. Yu, X., Carroll, S., Rigaud, J. L. & Inesi, G. H. + countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes. Biophys. J. 64, 1232–1242 (1993)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405, 647–655 (2000)

    CAS  ADS  Article  PubMed  Google Scholar 

  10. Toyoshima, C. & Nomura, H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605–611 (2002)

    CAS  ADS  Article  PubMed  Google Scholar 

  11. Sorensen, T. L., Moller, J. V. & Nissen, P. Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304, 1672–1675 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  12. Toyoshima, C. & Mizutani, T. Crystal structure of the calcium pump with a bound ATP analogue. Nature 430, 529–535 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  13. Toyoshima, C., Nomura, H. & Tsuda, T. Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature 432, 361–368 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  14. Olesen, C., Sorensen, T. L., Nielsen, R. C., Moller, J. V. & Nissen, P. Dephosphorylation of the calcium pump coupled to counterion occlusion. Science 306, 2251–2255 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  15. Jensen, A. M., Sorensen, T. L., Olesen, C., Moller, J. V. & Nissen, P. Modulatory and catalytic modes of ATP binding by the calcium pump. EMBO J. 25, 2305–2314 (2006)

    Article  PubMed  PubMed Central  Google Scholar 

  16. Feher, J. J. & Briggs, F. N. Determinants of calcium loading at steady state in sarcoplasmic reticulum. Biochim. Biophys. Acta 727, 389–402 (1983)

    CAS  Article  PubMed  Google Scholar 

  17. Gerdes, U. & Moller, J. V. The Ca2+ permeability of sarcoplasmic reticulum vesicles. II. Ca2+ efflux in the energized state of the calcium pump. Biochim. Biophys. Acta 734, 191–200 (1983)

    CAS  Article  PubMed  Google Scholar 

  18. Yu, X. & Inesi, G. Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase. J. Biol. Chem. 270, 4361–4367 (1995)

    CAS  Article  PubMed  Google Scholar 

  19. Artigas, P. & Gadsby, D. C. Na+/K+-pump ligands modulate gating of palytoxin-induced ion channels. Proc. Natl Acad. Sci. USA 100, 501–505 (2003)

    CAS  ADS  Article  PubMed  Google Scholar 

  20. Tanford, C. Translocation pathway in the catalysis of active transport. Proc. Natl Acad. Sci. USA 80, 3701–3705 (1983)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  21. Toyoshima, C. & Inesi, G. Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 73, 269–292 (2004)

    CAS  Article  PubMed  Google Scholar 

  22. Takahashi, M., Kondou, Y. & Toyoshima, C. Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors. Proc. Natl Acad. Sci. USA 104, 5800–5805 (2007)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  23. Taylor, J. S. Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5′-yl imidodiphosphate. J. Biol. Chem. 256, 9793–9795 (1981)

    CAS  PubMed  Google Scholar 

  24. Meltzer, S. & Berman, M. C. Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum. J. Biol. Chem. 259, 4244–4253 (1984)

    CAS  PubMed  Google Scholar 

  25. Mahaney, J. E., Thomas, D. D. & Froehlich, J. P. The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model. Biochemistry 43, 4400–4416 (2004)

    CAS  Article  PubMed  Google Scholar 

  26. Skou, J. C. The Na,K-pump. Methods Enzymol. 156, 1–25 (1988)

    CAS  Article  PubMed  Google Scholar 

  27. Läuger, P. in Electrogenic pumps Ch. 8 (Sinauer Associates, Sunderland, Massachusetts, 1991)

    Google Scholar 

  28. Danko, S., Yamasaki, K., Daiho, T. & Suzuki, H. Distinct natures of beryllium fluoride-bound, aluminum fluoride-bound, and magnesium fluoride-bound stable analogues of an ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase: changes in catalytic and transport sites during phosphoenzyme hydrolysis. J. Biol. Chem. 279, 14991–14998 (2004)

    CAS  Article  PubMed  Google Scholar 

  29. Picard, M., Toyoshima, C. & Champeil, P. Effects of inhibitors on luminal opening of Ca2+ binding sites in an E2P-like complex of the sarcoplasmic reticulum Ca2+-ATPase with Be2+-fluoride. J. Biol. Chem. 281, 3360–3369 (2006)

    CAS  Article  PubMed  Google Scholar 

  30. Moller, J. V. et al. Calcium transport by sarcoplasmic reticulum Ca2+-ATPase. Role of the A domain and its C-terminal link with the transmembrane region. J. Biol. Chem. 277, 38647–38659 (2002)

    CAS  Article  PubMed  Google Scholar 

  31. Daiho, T. et al. Deletions of any single residues in Glu40–Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate. J. Biol. Chem. 278, 39197–39204 (2003)

    CAS  Article  PubMed  Google Scholar 

  32. Lenoir, G. et al. Functional properties of sarcoplasmic reticulum Ca2+-ATPase after proteolytic cleavage at Leu119–Lys120, close to the A-domain. J. Biol. Chem. 279, 9156–9166 (2004)

    CAS  Article  PubMed  Google Scholar 

  33. Daiho, T., Yamasaki, K., Danko, S. & Suzuki, H. Critical role of Glu40–Ser48 loop linking actuator domain and 1st transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme. J. Biol. Chem. 282, 34429–34447 (2007)

    CAS  Article  PubMed  Google Scholar 

  34. Andersen, J. P. & Vilsen, B. Structure–function relationships of cation translocation by Ca2+- and Na+, K+-ATPases studied by site-directed mutagenesis. FEBS Lett. 359, 101–106 (1995)

    CAS  Article  PubMed  Google Scholar 

  35. Vilsen, B. & Andersen, J. P. Mutation to the glutamate in the fourth membrane segment of Na+,K+-ATPase and Ca2+-ATPase affects cation binding from both sides of the membrane and destabilizes the occluded enzyme forms. Biochemistry 37, 10961–10971 (1998)

    CAS  Article  PubMed  Google Scholar 

  36. Morth, J. P. et al. Crystal structure of the sodium–potassium pump. Nature doi: 10.1038/nature06419 (this issue)

  37. Reyes, N. & Gadsby, D. C. Ion permeation through the Na+,K+-ATPase. Nature 443, 470–474 (2006)

    CAS  ADS  Article  PubMed  Google Scholar 

  38. Wakabayashi, S., Ogurusu, T. & Shigekawa, M. Factors influencing calcium release from the ADP-sensitive phosphoenzyme intermediate of the sarcoplasmic reticulum ATPase. J. Biol. Chem. 261, 9762–9769 (1986)

    CAS  PubMed  Google Scholar 

  39. Apell, H. J. Structure–function relationship in P-type ATPases–a biophysical approach. Rev. Physiol. Biochem. Pharmacol. 150, 1–35 (2003)

    CAS  PubMed  Google Scholar 

  40. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966)

    CAS  ADS  Article  PubMed  Google Scholar 

  41. Vidaver, G. A. Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier. J. Theor. Biol. 10, 301–306 (1966)

    CAS  Article  PubMed  Google Scholar 

  42. Inesi, G., Ma, H., Lewis, D. & Xu, C. Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate. J. Biol. Chem. 279, 31629–31637 (2004)

    CAS  Article  PubMed  Google Scholar 

  43. Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli . Science 301, 610–615 (2003)

    CAS  ADS  Article  PubMed  Google Scholar 

  44. Boudker, O., Ryan, R. M., Yernool, D., Shimamoto, K. & Gouaux, E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387–393 (2007)

    CAS  ADS  Article  PubMed  Google Scholar 

  45. Dawson, R. J. & Locher, K. P. Structure of a bacterial multidrug ABC transporter. Nature 443, 180–185 (2006)

    CAS  ADS  Article  PubMed  Google Scholar 

  46. Hvorup, R. N. et al. Asymmetry in the structure of the ABC transporter–binding protein complex BtuCD–BtuF. Science 317, 1387–1390 (2006)

    ADS  Article  Google Scholar 

  47. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  PubMed  Google Scholar 

  48. Collet, J. F., Stroobant, V. & Van Schaftingen, E. Evidence for phosphotransferases phosphorylated on aspartate residue in N-terminal DXDX(T/V) motif. Methods Enzymol. 354, 177–188 (2002)

    CAS  Article  PubMed  Google Scholar 

  49. Purich, D. L. Use of sodium borohydride to detect acyl-phosphate linkages in enzyme reactions. Methods Enzymol. 354, 168–177 (2002)

    CAS  Article  PubMed  Google Scholar 

  50. Fiske, C. H. & Subbarow, Y. The colorimetric determination of phosphours. J. Biol. Chem. 26, 375–400 (1925)

    Google Scholar 

  51. Andersen, J. P., Lassen, K. & Moller, J. V. Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum. J. Biol. Chem. 260, 371–380 (1985)

    CAS  PubMed  Google Scholar 

  52. Sorensen, T. L., Olesen, C., Jensen, A. M., Moller, J. V. & Nissen, P. Crystals of sarcoplasmic reticulum Ca2+-ATPase. J. Biotechnol. 124, 704–716 (2006)

    CAS  Article  PubMed  Google Scholar 

  53. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    CAS  Article  Google Scholar 

  54. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  PubMed  Google Scholar 

  55. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  PubMed  Google Scholar 

  57. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    CAS  Article  PubMed  Google Scholar 

  58. Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993)

    CAS  Article  PubMed  Google Scholar 

  59. Sickmann, A. & Meyer, H. E. Phosphoamino acid analysis. Proteomics 1, 200–206 (2001)

    CAS  Article  PubMed  Google Scholar 

  60. Allegrini, S. et al. Bovine cytosolic 5′-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate. J. Biol. Chem. 276, 33526–33532 (2001)

    CAS  Article  PubMed  Google Scholar 

  61. Collet, J. F., Stroobant, V. & Van Schaftingen, E. Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases. J. Biol. Chem. 274, 33985–33990 (1999)

    CAS  Article  PubMed  Google Scholar 

  62. Sanders, D. A., Gillece-Castro, B. L., Stock, A. M., Burlingame, A. L. & Koshland, D. E. Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J. Biol. Chem. 264, 21770–21778 (1989)

    CAS  PubMed  Google Scholar 

  63. Murphy, A. J. & Coll, R. J. Fluoride is a slow, tight-binding inhibitor of the calcium ATPase of sarcoplasmic reticulum. J. Biol. Chem. 267, 5229–5235 (1992)

    CAS  PubMed  Google Scholar 

  64. Moller, J. V., Lind, K. E. & Andersen, J. P. Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein–protein interactions. J. Biol. Chem. 255, 1912–1920 (1980)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We dedicate this paper to the memory of B. Holm. We thank B. Nielsen, M.-B. Hemmingsen and A. M. Nielsen for technical assistance; J. L. Karlsen and F. Fredslund for technical discussions; and D. Flot and L. Gordon at beamlines ID 23-1 and -2 (operated jointly with EMBL-Grenoble) and ID 29 at the European Synchrotron Radiation Facility (ESRF) for help with data collection. Beamtime at the EMBL-DESY synchrotron Hamburg Germany is also acknowledged. This work was supported by the Danish Natural Science Research Council through the DANSYNC program, the Danish Medical Research Council, the Aarhus University Research Foundation, and the Novo Nordisk Foundation. C.Ol. is the recipient of a stipend from the PC Petersen Foundation and a PhD fellowship from the faculty of Health Sciences Aarhus University. A PhD fellowship (A.-M.L.W.) was financed by the Lundbeck Foundation. M.P. was supported by a post-doctoral fellowship from the Federation of European Biochemical Societies (FEBS) and P.N. is supported by a Hallas-Møller stipend of the Novo Nordisk Foundation.

Author Contributions C.Ol., M.P., A.-M.L.W., J.V.M. and P.N. contributed equally to this work. J.P.M. assisted with data collection and structure determination. C.Ox. and C.G. contributed with mass-spectrometry data and analysis.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Jesper Vuust Møller or Poul Nissen.

Additional information

The structural data have been deposited with the following codes in the Protein Data Bank: Ca2E1P, 3BA6; E2-AlF4-, 3B9R; and E2-BeF3-, 3B9B.

Supplementary information

Supplementary Information

The file contains Supplementary Table 1, Supplementary Figures S1-S6 with Legends and additional references. (PDF 1784 kb)

Supplementary Movie

The file contains Supplementary Movie 1. The movie shows the structure of the α β γ-complex of the Na,K-ATPase from pig kidney rotating (α-subunit in blue, β in wheat, γ in red, C-terminal switch in pink, and two Rb+ ions in magenta. The β ectodomain is represented by the experimental electron density (MOV 3297 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Olesen, C., Picard, M., Winther, AM. et al. The structural basis of calcium transport by the calcium pump. Nature 450, 1036–1042 (2007). https://doi.org/10.1038/nature06418

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06418

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing