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Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state

Nature volume 502, pages 201–206 (2013)Cite this article

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Abstract

Na+,K+-ATPase pumps three Na+ ions out of cells in exchange for two K+ taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na+ and K+ gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na+, ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na+ occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na+ ions are occluded. Details of the Na+-binding sites show how this ATPase functions as a Na+-specific pump, rejecting K+ and Ca2+, even though its affinity for Na+ is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na+ binding can now be formulated in atomic detail.

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Figure 1: Crystal structure of Na+,K+-ATPase in the transition state analogue E1∼P·ADP·3Na+.
Figure 2: Comparison of the Na+-bound form of Na+,K+-ATPase with a K+ bound form and with SERCA1a in the corresponding state.
Figure 3: Transmembrane Na+-binding sites.
Figure 4: A proposed model for binding sequence of three Na+.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures are deposited in the Protein Data Bank under accession numbers 3WGV (with oligomycin) and 3WGU (without oligomycin).

References

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

    Article  CAS  PubMed  Google Scholar 

  2. 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  Google Scholar 

  3. Apell, H. J. & Karlish, S. J. Functional properties of Na,K-ATPase, and their structural implications, as detected with biophysical techniques. J. Membr. Biol. 180, 1–9 (2001)

    Article  CAS  PubMed  Google Scholar 

  4. Morth, J. P. et al. Crystal structure of the sodium–potassium pump. Nature 450, 1043–1049 (2007)

    Article  CAS  ADS  PubMed  Google Scholar 

  5. Shinoda, T., Ogawa, H., Cornelius, F. & Toyoshima, C. Crystal structure of the sodium–potassium pump at 2.4 Å resolution. Nature 459, 446–450 (2009)

    Article  CAS  ADS  PubMed  Google Scholar 

  6. Ogawa, H., Shinoda, T., Cornelius, F. & Toyoshima, C. Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain. Proc. Natl Acad. Sci. USA 106, 13742–13747 (2009)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  7. Laursen, M., Yatime, L., Nissen, P. & Fedosova, N. U. Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site. Proc. Natl Acad. Sci. USA 110, 10958–10963 (2013)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  8. Toyoshima, C., Kanai, R. & Cornelius, F. First crystal structures of Na+,K+-ATPase: new light on the oldest ion pump. Structure 19, 1732–1738 (2011)

    Article  CAS  PubMed  Google Scholar 

  9. Morth, J. P. et al. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps. Nature Rev. Mol. Cell Biol. 12, 60–70 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

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

    Article  ADS  PubMed  Google Scholar 

  12. Esmann, M. & Skou, J. C. Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin. Biochem. Biophys. Res. Commun. 127, 857–863 (1985)

    Article  CAS  PubMed  Google Scholar 

  13. Glynn, I. M. in The Enzymes of Biological Membranes Vol. 3 (ed. A. N. Martonosi ) 35–114 (1985). New York and London: Plenum

  14. Abe, K., Tani, K., Nishizawa, T. & Fujiyoshi, Y. Inter-subunit interaction of gastric H+,K+-ATPase prevents reverse reaction of the transport cycle. EMBO J. 28, 1637–1643 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jacobsen, M. D., Pedersen, P. A. & Jørgensen, P. L. Importance of Na,K-ATPase residue α1-Arg544 in the segment Arg544–Asp567 for high-affinity binding of ATP, ADP, or MgATP. Biochemistry 41, 1451–1456 (2002)

    Article  CAS  PubMed  Google Scholar 

  16. Nayal, M. & Di Cera, E. Valence screening of water in protein crystals reveals potential Na+ binding sites. J. Mol. Biol. 256, 228–234 (1996)

    Article  CAS  PubMed  Google Scholar 

  17. Pintschovius, J., Fendler, K. & Bamberg, E. Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release. Biophys. J. 76, 827–836 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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)

    Article  CAS  ADS  PubMed  Google Scholar 

  19. Guàrdia, E., Rey, R. & Padró, J. A. Na+–Na+ and Cl−–Cl− ion pairs in water: mean force potentials by constrained molecular dynamics. J. Chem. Phys. 95, 2823–2831 (1991)

    Article  ADS  Google Scholar 

  20. Vilsen, B. Mutant Glu781→Ala of the rat kidney Na+,K+-ATPase displays low cation affinity and catalyzes ATP hydrolysis at a high rate in the absence of potassium ions. Biochemistry 34, 1455–1463 (1995)

    Article  CAS  PubMed  Google Scholar 

  21. Vilsen, B., Ramlov, D. & Andersen, J. P. Functional consequences of mutations in the transmembrane core region for cation translocation and energy transduction in the Na+,K+-ATPase and the SR Ca2+-ATPase. Ann. NY Acad. Sci. 834, 297–309 (1997)

    Article  CAS  ADS  PubMed  Google Scholar 

  22. Pedersen, P. A., Nielsen, J. M., Rasmussen, J. H. & Jørgensen, P. L. Contribution to Tl+, K+, and Na+ binding of Asn776, Ser775, Thr774, Thr772, and Tyr771 in cytoplasmic part of fifth transmembrane segment in α-subunit of renal Na,K-ATPase. Biochemistry 37, 17818–17827 (1998)

    Article  CAS  PubMed  Google Scholar 

  23. Kuntzweiler, T. A., Arguello, J. M. & Lingrel, J. B. Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase α subunit are cation coordinating residues. J. Biol. Chem. 271, 29682–29687 (1996)

    Article  CAS  PubMed  Google Scholar 

  24. Imagawa, T., Yamamoto, T., Kaya, S., Sakaguchi, K. & Taniguchi, K. Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity. J. Biol. Chem. 280, 18736–18744 (2005)

    Article  CAS  PubMed  Google Scholar 

  25. Einholm, A. P., Toustrup-Jensen, M. S., Holm, R., Andersen, J. P. & Vilsen, B. The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase α3 isoform disrupts Na+ interaction at the third Na+ site. J. Biol. Chem. 285, 26245–26254 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Morth, J. P. et al. The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations. Phil. Trans. R. Soc. B 364, 217–227 (2009)

    Article  CAS  PubMed  Google Scholar 

  27. Schack, V. R., Holm, R. & Vilsen, B. Inhibition of phosphorylation of Na+,K+-ATPase by mutations causing familial hemiplegic migraine. J. Biol. Chem. 287, 2191–2202 (2012)

    Article  CAS  PubMed  Google Scholar 

  28. Jewell-Motz, E. A. & Lingrel, J. B. Site-directed mutagenesis of the Na,K-ATPase: consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains. Biochemistry 32, 13523–13530 (1993)

    Article  CAS  PubMed  Google Scholar 

  29. Vilsen, B. Glutamate 329 located in the fourth transmembrane segment of the α-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions. Biochemistry 32, 13340–13349 (1993)

    Article  CAS  PubMed  Google Scholar 

  30. Clarke, D. M., Loo, T. W., Inesi, G. & MacLennan, D. H. Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature 339, 476–478 (1989)

    Article  CAS  ADS  PubMed  Google Scholar 

  31. Toustrup-Jensen, M. S. et al. The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935. J. Biol. Chem. 284, 18715–18725 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yaragatupalli, S., Olivera, J. F., Gatto, C. & Artigas, P. Altered Na+ transport after an intracellular α-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump. Proc. Natl Acad. Sci. USA 106, 15507–15512 (2009)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  33. Meier, S., Tavraz, N. N., Durr, K. L. & Friedrich, T. Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit. J. Gen. Physiol. 135, 115–134 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vedovato, N. & Gadsby, D. C. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions. J. Gen. Physiol. 136, 63–82 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schulz, S. & Apell, H. J. Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump. Eur. Biophys. J. 23, 413–421 (1995)

    Article  CAS  PubMed  Google Scholar 

  36. Cornelius, F. & Skou, J. C. The sided action of Na+ on reconstituted shark Na+/K+-ATPase engaged in Na+–Na+ exchange accompanied by ATP hydrolysis. II. Transmembrane allosteric effects on Na+ affinity. Biochim. Biophys. Acta 944, 223–232 (1988)

    Article  CAS  PubMed  Google Scholar 

  37. Schneeberger, A. & Apell, H. J. Ion selectivity of the cytoplasmic binding sites of the Na,K-ATPase: II. Competition of various cations. J. Membr. Biol. 179, 263–273 (2001)

    Article  CAS  PubMed  Google Scholar 

  38. Toyoshima, C. et al. Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state. Nature 495, 260–264 (2013)

    Article  CAS  ADS  PubMed  Google Scholar 

  39. Winther, A. M. et al. The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 495, 265–269 (2013)

    Article  CAS  ADS  PubMed  Google Scholar 

  40. Jørgensen, P. L. Purification and characterization of (Na+ + K+)-ATPase. 3. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim. Biophys. Acta 356, 36–52 (1974)

    Article  PubMed  Google Scholar 

  41. Schack, V. R. et al. Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase. J. Biol. Chem. 283, 27982–27990 (2008)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  44. Fabiola, F., Bertram, R., Korostelev, A. & Chapman, M. S. An improved hydrogen bond potential: impact on medium resolution protein structures. Protein Sci. 11, 1415–1423 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  47. McDonald, I. K. & Thornton, J. M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Brown, I. D. & Wu, K. K. Empirical parameters for calculating cation-oxygen bond valences. Acta Crystallogr. B 32, 1957–1959 (1976)

    Article  Google Scholar 

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

    Article  Google Scholar 

  51. Ho, B. K. & Gruswitz, F. HOLLOW: Generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Hasegawa and H. Okumura of the Japan Synchrotron Radiation Research Institute (JASRI) for data collection at BL41XU of SPring-8, and J. Petersen, A. M. Beck Sørensen and A. Lillevang for technical assistance with membrane purification. We are grateful to D. B. McIntosh for help in improving the manuscript, and J. Tsueda for help in preparing figures. This work is a part of a long-term project (2009B0025) at SPring-8 and was supported by a Specially Promoted Project Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.T.) as well as by research grants from the Lundbeck Foundation (to B.V.), the Novo Nordisk Foundation, Fabrikant Vilhelm Pedersen og Hustrus Legat (to B.V. and F.C.), the Danish Medical Research Council (to B.V. and F.C.) and the FI/Danish-Japanese cooperation program from the Danish Agency for Science Technology and Innovation (to F.C.).

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  1. Ryuta Kanai and Haruo Ogawa: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan,

    Ryuta Kanai, Haruo Ogawa & Chikashi Toyoshima

  2. Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark,

    Bente Vilsen & Flemming Cornelius

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  1. Ryuta Kanai
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Contributions

C.T., B.V. and F.C. planned and supervised the study. B.V. and F.C. prepared purified membranes containing Na+,K+-ATPase. R.K. and C.T. crystallized the ATPase; R.K., H.O. and C.T. collected diffraction data, determined the structure and prepared figures. C.T., B.V. and F.C. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Chikashi Toyoshima.

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Kanai, R., Ogawa, H., Vilsen, B. et al. Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state. Nature 502, 201–206 (2013). https://doi.org/10.1038/nature12578

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