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Structural changes in the calcium pump accompanying the dissociation of calcium

Abstract

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca2+ ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 Å resolution in a Ca2+-free (E2) state, and compare it with that determined previously for the Ca2+-bound (E1Ca2+) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca2+. Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.

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Figure 1: Ribbon representation of SR Ca2+-ATPase in the Ca2+-bound form (E1Ca2+) and that (E2(TG)) in the absence of Ca2+ but in the presence of thapsigargin (TG).
Figure 2: Superimposition of the Ca2+-bound form (E1Ca2+, violet) and the thapsigargin-bound form (E2(TG), light green) of Ca2+-ATPase fitted with the transmembrane domain. α-Helices are represented by cylinders and β-strands by arrows.
Figure 3: Interface between the transmembrane helices (M3–M5) and the P domain of Ca2+-ATPase.
Figure 4: Rearrangement of transmembrane helices viewed from the rear (a), and a diagram illustrating the shift of M4 normal to the membrane by the tilting of M5 (b).
Figure 5: Conformation changes around the Ca2+-binding sites.
Figure 6: Thapsigargin (TG) binding site in Ca2+-ATPase.

References

  1. Møller, J. V., Juul, B. & le Maire, M. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1–51 (1996)

    PubMed  Article  Google Scholar 

  2. MacLennan, D. H., Rice, W. J. & Green, N. M. The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases. J. Biol. Chem. 272, 28815–28818 (1997)

    CAS  PubMed  Article  Google Scholar 

  3. Lee, A. G. & East, J. M. What the structure of a calcium pump tells us about its mechanism. Biochem. J. 356, 665–683 (2001)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 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  PubMed  PubMed Central  Article  Google Scholar 

  5. Post, R. L., Hegyvary, C. & Kume, S. Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 247, 6530–6540 (1972)

    CAS  PubMed  Article  Google Scholar 

  6. Albers, R. W. Biochemical aspects of active transport. Annu. Rev. Biochem. 36, 727–756 (1967)

    CAS  PubMed  Article  Google Scholar 

  7. de Meis, L. & Vianna, A. L. Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 48, 275–292 (1979)

    CAS  PubMed  Article  Google Scholar 

  8. Aravind, L., Galperin, M. Y. & Koonin, E. V. The catalytic domain of the P-type ATPase has the haloacid dehalogenase fold. Trends Biochem. Sci. 23, 127–129 (1998)

    CAS  PubMed  Article  Google Scholar 

  9. Johnson, L. N. & Lewis, R. J. Structural basis for control by phosphorylation. Chem. Rev. 101, 2209–2242 (2001)

    CAS  PubMed  Article  Google Scholar 

  10. 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)

    ADS  CAS  PubMed  Article  Google Scholar 

  11. Xu, C., Rice, W. J., He, W. & Stokes, D. L. A structural model for the catalytic cycle of Ca2+-ATPase. J. Mol. Biol. 316, 201–211 (2002)

    CAS  PubMed  Article  Google Scholar 

  12. Zhang, P., Toyoshima, C., Yonekura, K., Green, N. M. & Stokes, D. L. Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution. Nature 392, 835–839 (1998)

    ADS  CAS  PubMed  Article  Google Scholar 

  13. Danko, S. et al. ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin. FEBS Lett. 489, 277–282 (2001)

    CAS  PubMed  Article  Google Scholar 

  14. Danko, S., Yamasaki, K., Daiho, T., Suzuki, H. & Toyoshima, C. Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+-ATPase in E1P and E1ATP states: a limited proteolysis study. FEBS Lett. 505, 129–135 (2001)

    CAS  PubMed  Article  Google Scholar 

  15. Sagara, Y. & Inesi, G. Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations. J. Biol. Chem. 266, 13503–13506 (1991)

    CAS  PubMed  Article  Google Scholar 

  16. Hayward, S. Structural principles governing domain motions in proteins. Proteins 36, 425–435 (1999)

    CAS  PubMed  Article  Google Scholar 

  17. Zhang, Z., Lewis, D., Sumbilla, C., Inesi, G. & Toyoshima, C. The role of the M6-M7 loop (L67) in stabilization of the phosphorylation and Ca2+ binding domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). J. Biol. Chem. 276, 15232–15239 (2001)

    CAS  PubMed  Article  Google Scholar 

  18. Zhang, Z. et al. Mutational analysis of the peptide segment linking phosphorylation and Ca2+-binding domains in the sarcoplasmic reticulum Ca2+-ATPase. J. Biol. Chem. 270, 16283–16290 (1995)

    CAS  PubMed  Article  Google Scholar 

  19. Andersen, J. P., Vilsen, B. & MacLennan, D. H. Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 267, 2767–2774 (1992)

    CAS  PubMed  Article  Google Scholar 

  20. Zhang, Z. et al. Detailed characterization of the cooperative mechanism of Ca2+ binding and catalytic activation in the Ca2+ transport (SERCA) ATPase. Biochemistry 39, 8758–8767 (2000)

    CAS  PubMed  Article  Google Scholar 

  21. Andersen, J. P. & Vilsen, B. Amino acids Asn796 and Thr799 of the Ca2+-ATPase of sarcoplasmic reticulum bind Ca2+ at different sites. J. Biol. Chem. 269, 15931–15936 (1994)

    CAS  PubMed  Article  Google Scholar 

  22. Glusker, J. P. Structural aspects of metal liganding to functional groups in proteins. Adv. Protein Chem. 42, 1–76 (1991)

    CAS  PubMed  Article  Google Scholar 

  23. Medda, P., Fassold, E. & Hasselbach, W. The effect of monovalent and divalent cations on the ATP-dependent Ca2+- binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase. Eur. J. Biochem. 165, 251–259 (1987)

    CAS  PubMed  Article  Google Scholar 

  24. Yu, M. et al. Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells. J. Biol. Chem. 273, 3542–3546 (1998)

    CAS  PubMed  Article  Google Scholar 

  25. Hua, S. & Inesi, G. Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase. Biochemistry 36, 11865–11872 (1997)

    CAS  PubMed  Article  Google Scholar 

  26. Pikula, S., Mullner, N., Dux, L. & Martonosi, A. Stabilization and crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum. J. Biol. Chem. 263, 5277–5286 (1988)

    CAS  PubMed  Article  Google Scholar 

  27. Juul, B. et al. Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca2+-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K? J. Biol. Chem. 270, 20123–20134 (1995)

    CAS  PubMed  Article  Google Scholar 

  28. Lutsenko, S., Anderko, R. & Kaplan, J. H. Membrane disposition of the M5-M6 hairpin of Na+, K+-ATPase α subunit is ligand dependent. Proc. Natl Acad. Sci. USA 92, 7936–7940 (1995)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Gatto, C., Lutsenko, S., Shin, J. M., Sachs, G. & Kaplan, J. H. Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for P2-type ATPases. J. Biol. Chem. 274, 13737–13740 (1999)

    CAS  PubMed  Article  Google Scholar 

  30. Juul, B. & Møller, J. V. in Na/K-ATPase and Related ATPases (eds Taniguchi, K. & Kaya, S.) 233–236 (Elsevier, Amsterdam, 2000)

    Google Scholar 

  31. Jørgensen, P. L. & Collins, J. H. Tryptic and chymotryptic cleavage sites in sequence of α-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney. Biochim. Biophys. Acta 860, 570–576 (1986)

    PubMed  Article  Google Scholar 

  32. Coll, R. J. & Murphy, A. J. Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography. J. Biol. Chem. 259, 14249–14254 (1984)

    CAS  PubMed  Article  Google Scholar 

  33. Stokes, D. L. & Green, N. M. Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys. J. 57, 1–14 (1990)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–325 (1997)

    CAS  Article  PubMed  Google Scholar 

  35. Brünger, A. T. Extension of molecular replacement: a new search strategy based on Patterson correlation refinement. Acta Crystallogr. A 46, 46–57 (1990)

    Article  Google Scholar 

  36. Collaborative Computational Project No. 4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

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

    PubMed  Article  Google Scholar 

  38. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983)

    CAS  PubMed  Article  Google Scholar 

  39. Kutschabsky, L., Kretschmer, R.-G. & Ripperger, H. The crystal and molecular structure of the sesquiterpenoid silerin (trilobolide). Crystal Res. Technol. 21, 627–633 (1986)

    CAS  Article  Google Scholar 

  40. Christensen, S. B., Larsen, I. K., Rasmussen, U. & Christopherson, C. Thapsigargin and thapsigargicin, two histamine liberating sesquiterpene lactones from Thapsia garganica. X-ray analysis of the 7,11-epoxide of thapsigargin. J. Org. Chem. 47, 649–652 (1982)

    Article  Google Scholar 

  41. MacLennan, D. H., Brandl, C. J., Korczak, B. & Green, N. M. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316, 696–700 (1985)

    ADS  CAS  PubMed  Article  Google Scholar 

  42. James, P., Inui, M., Tada, M., Chiesi, M. & Carafoli, E. Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature 342, 90–92 (1989)

    ADS  CAS  PubMed  Article  Google Scholar 

  43. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)

    Article  Google Scholar 

  44. Christensen, S. B., Andersen, A. & Smitt, U. W. Sesquiterpenoids from Thapsia species and medicinal chemistry of the thapsigargins. Fort. Chem. Org. Nat. 71, 129–167 (1997)

    CAS  Google Scholar 

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Acknowledgements

We thank H. Ogawa for help in data gathering, R. Yoshida for computations, and M. Nakasako for modelling. We also thank G. Inesi, P. Champeil, D. B. McIntosh and H. Suzuki for communicating unpublished results to us and for their help in improving the manuscript. Thanks are also due to E. Yamashita and all the staff at BL44XU of SPring-8. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japan New Energy and Industry Technology Development Organization, and the Human Frontier Science Program.

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Correspondence to Chikashi Toyoshima.

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Toyoshima, C., Nomura, H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605–611 (2002). https://doi.org/10.1038/nature00944

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