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. 2002 May 15;364(Pt 1):293–299. doi: 10.1042/bj3640293

Role of the self-association of beta subunits in the oligomeric structure of Na+/K+-ATPase.

Alexander V Ivanov 1, Nikolai N Modyanov 1, Amir Askari 1
PMCID: PMC1222572  PMID: 11988103

Abstract

The two subunits of Na(+)/K(+)-ATPase that are essential for function are alpha and beta. Previous cross-linking studies on the oligomeric structure of the membrane-bound enzyme identified alpha,beta and alpha,alpha associations, but only the former and not the latter could be detected after solubilization. To study the possibility of direct beta,beta association, the purified membrane enzyme and a trypsin-digested enzyme that occludes cations and contains an essentially intact beta and fragments of alpha were subjected to oxidative cross-linking in the presence of Cu(2+)-phenanthroline. Resolution of products on polyacrylamide gels, N-terminal analysis and reactivity with anti-beta antibody showed that, in addition to previously identified products (e.g. alpha,alpha and alpha,beta dimers), a beta,beta dimer, most likely linked through intramembrane Cys(44) residues of two chains, is also formed. This dimer was also noted when digitonin-solubilized intact enzyme, and the trypsin-digested enzyme solubilized with digitonin or polyoxyethylene 10-laurylether were subjected to cross-linking, indicating that the detected beta,beta association was not due to random collisions. In the digested enzyme, K(+) but not Na(+) enhanced beta,beta dimer formation. The alternative cross-linking of beta-Cys(44) to a Cys residue of a transmembrane alpha-helix was antagonized specifically by K(+) or Na(+). The findings (i) indicate the role of beta,beta association in maintaining the minimum oligomeric structure of (alpha,beta)(2), (ii) provide further support for conformation-dependent flexibilities of the spatial relations of the transmembrane helices of alpha and beta and (iii) suggest the possibility of significant differences between the quaternary structures of the P-type ATPases that do and do not contain a beta subunit.

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Selected References

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  1. Andersen J. P. Monomer-oligomer equilibrium of sarcoplasmic reticulum Ca-ATPase and the role of subunit interaction in the Ca2+ pump mechanism. Biochim Biophys Acta. 1989 Jan 18;988(1):47–72. doi: 10.1016/0304-4157(89)90003-8. [DOI] [PubMed] [Google Scholar]
  2. Antolovic R., Hamer E., Serpersu E. H., Kost H., Linnertz H., Kovarik Z., Schoner W. Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase. Eur J Biochem. 1999 Apr;261(1):181–189. doi: 10.1046/j.1432-1327.1999.00260.x. [DOI] [PubMed] [Google Scholar]
  3. Arystarkhova E., Wetzel R. K., Asinovski N. K., Sweadner K. J. The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase. J Biol Chem. 1999 Nov 19;274(47):33183–33185. doi: 10.1074/jbc.274.47.33183. [DOI] [PubMed] [Google Scholar]
  4. Askari A. (Na+ + K+)-ATPase: on the number of the ATP sites of the functional unit. J Bioenerg Biomembr. 1987 Aug;19(4):359–374. doi: 10.1007/BF00768539. [DOI] [PubMed] [Google Scholar]
  5. Askari A., Huang W. H., McCormick P. W. (Na+ + K+)-dependent adenosine triphosphatase. Regulation of inorganic phosphate, magnesium ion, and calcium ion interactions with the enzyme by ouabain. J Biol Chem. 1983 Mar 25;258(6):3453–3460. [PubMed] [Google Scholar]
  6. Askari A., Huang W., Antieau J. M. Na+,K+-ATPase: ligand-induced conformational transitions and alterations in subunit interactions evidenced by cross-linking studies. Biochemistry. 1980 Mar 18;19(6):1132–1140. doi: 10.1021/bi00547a015. [DOI] [PubMed] [Google Scholar]
  7. Askari A., Huang W. Na+,K+-ATPase: half-of-the-subunits cross-linking reactivity suggests an oligomeric structure containing a minimum of four catalytic subunits. Biochem Biophys Res Commun. 1980 Mar 28;93(2):448–453. doi: 10.1016/0006-291x(80)91098-0. [DOI] [PubMed] [Google Scholar]
  8. Askari A. Overview: ligand binding sites of (Na+ + K+)-ATPase: nucleotides and cations. Prog Clin Biol Res. 1988;268A:149–165. [PubMed] [Google Scholar]
  9. Boldyrev A. A. Na/K-ATPase as an oligomeric ensemble. Biochemistry (Mosc) 2001 Aug;66(8):821–831. doi: 10.1023/a:1011964832767. [DOI] [PubMed] [Google Scholar]
  10. Béguin P., Wang X., Firsov D., Puoti A., Claeys D., Horisberger J. D., Geering K. The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function. EMBO J. 1997 Jul 16;16(14):4250–4260. doi: 10.1093/emboj/16.14.4250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Capasso J. M., Hoving S., Tal D. M., Goldshleger R., Karlish S. J. Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites. J Biol Chem. 1992 Jan 15;267(2):1150–1158. [PubMed] [Google Scholar]
  12. Craig W. S., Kyte J. Stoichiometry and molecular weight of the minimum asymmetric unit of canine renal sodium and potassium ion-activated adenosine triphosphatase. J Biol Chem. 1980 Jul 10;255(13):6262–6269. [PubMed] [Google Scholar]
  13. Donnet C., Arystarkhova E., Sweadner K. J. Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain. J Biol Chem. 2000 Nov 30;276(10):7357–7365. doi: 10.1074/jbc.M009131200. [DOI] [PubMed] [Google Scholar]
  14. Froehlich J. P., Taniguchi K., Fendler K., Mahaney J. E., Thomas D. D., Albers R. W. Complex kinetic behavior in the Na,K- and Ca-ATPases. Evidence for subunit-subunit interactions and energy conservation during catalysis. Ann N Y Acad Sci. 1997 Nov 3;834:280–296. doi: 10.1111/j.1749-6632.1997.tb52259.x. [DOI] [PubMed] [Google Scholar]
  15. Ganjeizadeh M., Zolotarjova N., Huang W. H., Askari A. Interactions of phosphorylation and dimerizing domains of the alpha-subunits of Na+/K(+)-ATPase. J Biol Chem. 1995 Jun 30;270(26):15707–15710. doi: 10.1074/jbc.270.26.15707. [DOI] [PubMed] [Google Scholar]
  16. Hasler U., Crambert G., Horisberger J. D., Geering K. Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning. J Biol Chem. 2001 Feb 13;276(19):16356–16364. doi: 10.1074/jbc.M008778200. [DOI] [PubMed] [Google Scholar]
  17. Huang W., Askari A. Na+,K+-ATPase: on the nature of the "cross-linked" subunits obtained in the presence of o-phenanthroline and cupric ion. Biochem Biophys Res Commun. 1978 Jun 29;82(4):1314–1319. doi: 10.1016/0006-291x(78)90331-5. [DOI] [PubMed] [Google Scholar]
  18. Ivanov A., Zhao H., Modyanov N. N. Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking. Biochemistry. 2000 Aug 15;39(32):9778–9785. doi: 10.1021/bi001004j. [DOI] [PubMed] [Google Scholar]
  19. Karlish S. J., Goldshleger R., Stein W. D. A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4566–4570. doi: 10.1073/pnas.87.12.4566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koster J. C., Blanco G., Mercer R. W. A cytoplasmic region of the Na,K-ATPase alpha-subunit is necessary for specific alpha/alpha association. J Biol Chem. 1995 Jun 16;270(24):14332–14339. doi: 10.1074/jbc.270.24.14332. [DOI] [PubMed] [Google Scholar]
  21. Kyte J. Molecular considerations relevant to the mechanism of active transport. Nature. 1981 Jul 16;292(5820):201–204. doi: 10.1038/292201a0. [DOI] [PubMed] [Google Scholar]
  22. Liang S. M., Winter C. G. Digitonin-induced changes in subunit arrangement in relation to some in vitro activities of the (Na+,K+)-ATPase. J Biol Chem. 1977 Nov 25;252(22):8278–8284. [PubMed] [Google Scholar]
  23. Liu G., Xie Z., Modyanov N. N., Askari A. Restoration of phosphorylation capacity to the dormant half of the alpha-subunits of Na+, K(+)-ATPase. FEBS Lett. 1996 Jul 29;390(3):323–326. doi: 10.1016/0014-5793(96)00687-4. [DOI] [PubMed] [Google Scholar]
  24. Lutsenko S., Kaplan J. H. An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion. Biochemistry. 1993 Jul 6;32(26):6737–6743. doi: 10.1021/bi00077a029. [DOI] [PubMed] [Google Scholar]
  25. Lutsenko S., Kaplan J. H. Organization of P-type ATPases: significance of structural diversity. Biochemistry. 1995 Dec 5;34(48):15607–15613. doi: 10.1021/bi00048a001. [DOI] [PubMed] [Google Scholar]
  26. MacKenzie K. R., Prestegard J. H., Engelman D. M. A transmembrane helix dimer: structure and implications. Science. 1997 Apr 4;276(5309):131–133. doi: 10.1126/science.276.5309.131. [DOI] [PubMed] [Google Scholar]
  27. Martin D. W., Sachs J. R. Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites. Biochemistry. 1999 Jun 8;38(23):7485–7497. doi: 10.1021/bi983019b. [DOI] [PubMed] [Google Scholar]
  28. Martonosi A. N. Structure-function relationships in the Ca(2+)-ATPase of sarcoplasmic reticulum: facts, speculations and questions for the future. Biochim Biophys Acta. 1996 Jul 18;1275(1-2):111–117. doi: 10.1016/0005-2728(96)00059-x. [DOI] [PubMed] [Google Scholar]
  29. Nørby J. G., Jensen J. Functional significance of the oligomeric structure of the Na,K-pump from radiation inactivation and ligand binding. Soc Gen Physiol Ser. 1991;46:173–188. [PubMed] [Google Scholar]
  30. Or E., Goldshleger E. D., Tal D. M., Karlish S. J. Solubilization of a complex of tryptic fragments of Na,K-ATPase containing occluded Rb ions and bound ouabain. Biochemistry. 1996 May 28;35(21):6853–6864. doi: 10.1021/bi960093q. [DOI] [PubMed] [Google Scholar]
  31. Or E., Goldshleger R., Karlish S. J. Characterization of disulfide cross-links between fragments of proteolyzed Na,K-ATPase. Implications for spatial organization of trans-membrane helices. J Biol Chem. 1999 Jan 29;274(5):2802–2809. doi: 10.1074/jbc.274.5.2802. [DOI] [PubMed] [Google Scholar]
  32. Periyasamy S. M., Huang W. H., Askari A. Subunit associations of (Na+ + K+)-dependent adenosine triphosphatase. Chemical cross-linking studies. J Biol Chem. 1983 Aug 25;258(16):9878–9885. [PubMed] [Google Scholar]
  33. Peters K., Richards F. M. Chemical cross-linking: reagents and problems in studies of membrane structure. Annu Rev Biochem. 1977;46:523–551. doi: 10.1146/annurev.bi.46.070177.002515. [DOI] [PubMed] [Google Scholar]
  34. Popot J. L., Engelman D. M. Helical membrane protein folding, stability, and evolution. Annu Rev Biochem. 2000;69:881–922. doi: 10.1146/annurev.biochem.69.1.881. [DOI] [PubMed] [Google Scholar]
  35. Sarvazyan N. A., Ivanov A., Modyanov N. N., Askari A. Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase. J Biol Chem. 1997 Mar 21;272(12):7855–7858. doi: 10.1074/jbc.272.12.7855. [DOI] [PubMed] [Google Scholar]
  36. Sarvazyan N. A., Modyanov N. N., Askari A. Intersubunit and intrasubunit contact regions of Na+/K(+)-ATPase revealed by controlled proteolysis and chemical cross-linking. J Biol Chem. 1995 Nov 3;270(44):26528–26532. doi: 10.1074/jbc.270.44.26528. [DOI] [PubMed] [Google Scholar]
  37. Shin J. M., Sachs G. Dimerization of the gastric H+, K(+)-ATPase. J Biol Chem. 1996 Jan 26;271(4):1904–1908. doi: 10.1074/jbc.271.4.1904. [DOI] [PubMed] [Google Scholar]
  38. Skou J. C., Esmann M. The Na,K-ATPase. J Bioenerg Biomembr. 1992 Jun;24(3):249–261. doi: 10.1007/BF00768846. [DOI] [PubMed] [Google Scholar]
  39. Steck T. L. Cross-linking the major proteins of the isolated erythrocyte membrane. J Mol Biol. 1972 May 14;66(2):295–305. doi: 10.1016/0022-2836(72)90481-0. [DOI] [PubMed] [Google Scholar]
  40. Sweadner K. J., Donnet C. Structural similarities of Na,K-ATPase and SERCA, the Ca(2+)-ATPase of the sarcoplasmic reticulum. Biochem J. 2001 Jun 15;356(Pt 3):685–704. doi: 10.1042/0264-6021:3560685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Taniguchi K., Kaya S., Abe K., Mårdh S. The oligomeric nature of Na/K-transport ATPase. J Biochem. 2001 Mar;129(3):335–342. doi: 10.1093/oxfordjournals.jbchem.a002862. [DOI] [PubMed] [Google Scholar]
  42. Thoenges D., Amler E., Eckert T., Schoner W. Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E2ATP site leads to inhibition of Na+/K+-ATPase. Analysis of structural requirements of fluorescent ATP derivatives with a Koshland-Némethy-Filmer model of two interacting ATP sites. J Biol Chem. 1999 Jan 22;274(4):1971–1978. doi: 10.1074/jbc.274.4.1971. [DOI] [PubMed] [Google Scholar]
  43. Toyoshima C., Nakasako M., Nomura H., Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature. 2000 Jun 8;405(6787):647–655. doi: 10.1038/35015017. [DOI] [PubMed] [Google Scholar]
  44. Tsuda T., Kaya S., Yokoyama T., Hayashi Y., Taniguchi K. ATP and acetyl phosphate induces molecular events near the ATP binding site and the membrane domain of Na+,K+-ATPase. The tetrameric nature of the enzyme. J Biol Chem. 1998 Sep 18;273(38):24339–24345. doi: 10.1074/jbc.273.38.24339. [DOI] [PubMed] [Google Scholar]
  45. Ward D. G., Cavieres J. D. Solubilized alpha beta Na,K-ATPase remains protomeric during turnover yet shows apparent negative cooperativity toward ATP. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5332–5336. doi: 10.1073/pnas.90.11.5332. [DOI] [PMC free article] [PubMed] [Google Scholar]

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