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. 1999 Apr;76(4):1784–1795. doi: 10.1016/S0006-3495(99)77339-X

Structure of the 1-36 amino-terminal fragment of human phospholamban by nuclear magnetic resonance and modeling of the phospholamban pentamer.

P Pollesello 1, A Annila 1, M Ovaska 1
PMCID: PMC1300156  PMID: 10096878

Abstract

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.

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Selected References

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  1. Adams P. D., Arkin I. T., Engelman D. M., Brünger A. T. Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban. Nat Struct Biol. 1995 Feb;2(2):154–162. doi: 10.1038/nsb0295-154. [DOI] [PubMed] [Google Scholar]
  2. Autry J. M., Jones L. R. Functional Co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation. J Biol Chem. 1997 Jun 20;272(25):15872–15880. doi: 10.1074/jbc.272.25.15872. [DOI] [PubMed] [Google Scholar]
  3. Baxter N. J., Williamson M. P. Temperature dependence of 1H chemical shifts in proteins. J Biomol NMR. 1997 Jun;9(4):359–369. doi: 10.1023/a:1018334207887. [DOI] [PubMed] [Google Scholar]
  4. Bechinger B. Structure and functions of channel-forming peptides: magainins, cecropins, melittin and alamethicin. J Membr Biol. 1997 Apr 1;156(3):197–211. doi: 10.1007/s002329900201. [DOI] [PubMed] [Google Scholar]
  5. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  6. Brünger A. T., Clore G. M., Gronenborn A. M., Karplus M. Three-dimensional structure of proteins determined by molecular dynamics with interproton distance restraints: application to crambin. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3801–3805. doi: 10.1073/pnas.83.11.3801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chazin W. J., Rance M., Wright P. E. Complete assignment of the 1H nuclear magnetic resonance spectrum of French bean plastocyanin. Application of an integrated approach to spin system identification in proteins. J Mol Biol. 1988 Aug 5;202(3):603–622. doi: 10.1016/0022-2836(88)90290-2. [DOI] [PubMed] [Google Scholar]
  8. Cornea R. L., Jones L. R., Autry J. M., Thomas D. D. Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers. Biochemistry. 1997 Mar 11;36(10):2960–2967. doi: 10.1021/bi961955q. [DOI] [PubMed] [Google Scholar]
  9. Dyson H. J., Merutka G., Waltho J. P., Lerner R. A., Wright P. E. Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. J Mol Biol. 1992 Aug 5;226(3):795–817. doi: 10.1016/0022-2836(92)90633-u. [DOI] [PubMed] [Google Scholar]
  10. Fujii J., Maruyama K., Tada M., MacLennan D. H. Expression and site-specific mutagenesis of phospholamban. Studies of residues involved in phosphorylation and pentamer formation. J Biol Chem. 1989 Aug 5;264(22):12950–12955. [PubMed] [Google Scholar]
  11. Fujii J., Ueno A., Kitano K., Tanaka S., Kadoma M., Tada M. Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban. J Clin Invest. 1987 Jan;79(1):301–304. doi: 10.1172/JCI112799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gao Y., Levine B. A., Mornet D., Slatter D. A., Strasburg G. M. Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy. Biochim Biophys Acta. 1992 Nov 10;1160(1):22–34. doi: 10.1016/0167-4838(92)90035-c. [DOI] [PubMed] [Google Scholar]
  13. Halkides C. J., Redfield A. G. The effect of 17O on the relaxation of an amide proton within a hydrogen bond. J Biomol NMR. 1995 Jun;5(4):362–366. doi: 10.1007/BF00182279. [DOI] [PubMed] [Google Scholar]
  14. Herzyk P., Hubbard R. E. Using experimental information to produce a model of the transmembrane domain of the ion channel phospholamban. Biophys J. 1998 Mar;74(3):1203–1214. doi: 10.1016/S0006-3495(98)77835-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hicks M. J., Shigekawa M., Katz A. M. Mechanism by which cyclic adenosine 3':5'-monophosphate-dependent protein kinase stimulates calcium transport in cardiac sarcoplasmic reticulum. Circ Res. 1979 Mar;44(3):384–391. doi: 10.1161/01.res.44.3.384. [DOI] [PubMed] [Google Scholar]
  16. Hubbard J. A., MacLachlan L. K., Meenan E., Salter C. J., Reid D. G., Lahouratate P., Humphries J., Stevens N., Bell D., Neville W. A. Conformation of the cytoplasmic domain of phospholamban by NMR and CD. Mol Membr Biol. 1994 Oct-Dec;11(4):263–269. doi: 10.3109/09687689409160436. [DOI] [PubMed] [Google Scholar]
  17. Hughes G., East J. M., Lee A. G. The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca(2+)-ATPase. Biochem J. 1994 Oct 15;303(Pt 2):511–516. doi: 10.1042/bj3030511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hughes G., Khan Y. M., East J. M., Lee A. G. Effects of polycations on Ca2+ binding to the Ca(2+)-ATPase. Biochem J. 1995 Jun 1;308(Pt 2):493–499. doi: 10.1042/bj3080493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hughes G., Starling A. P., East J. M., Lee A. G. Mechanism of inhibition of the Ca(2+)-ATPase by spermine and other polycationic compounds. Biochemistry. 1994 Apr 26;33(16):4745–4754. doi: 10.1021/bi00182a001. [DOI] [PubMed] [Google Scholar]
  20. Hughes G., Starling A. P., Sharma R. P., East J. M., Lee A. G. An investigation of the mechanism of inhibition of the Ca(2+)-ATPase by phospholamban. Biochem J. 1996 Sep 15;318(Pt 3):973–979. doi: 10.1042/bj3180973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Imagawa T., Watanabe T., Nakamura T. Subunit structure and multiple phosphorylation sites of phospholamban. J Biochem. 1986 Jan;99(1):41–53. doi: 10.1093/oxfordjournals.jbchem.a135478. [DOI] [PubMed] [Google Scholar]
  22. Iwasa T., Inoue N., Miyamoto E. Identification of a calmodulin-dependent protein kinase in the cardiac cytosol, which phosphorylates phospholamban in the sarcoplasmic reticulum. J Biochem. 1985 Aug;98(2):577–580. doi: 10.1093/oxfordjournals.jbchem.a135313. [DOI] [PubMed] [Google Scholar]
  23. Jackson W. A., Colyer J. Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca 2+-pump stimulation. Biochem J. 1996 May 15;316(Pt 1):201–207. doi: 10.1042/bj3160201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones L. R., Field L. J. Residues 2-25 of phospholamban are insufficient to inhibit Ca2+ transport ATPase of cardiac sarcoplasmic reticulum. J Biol Chem. 1993 Jun 5;268(16):11486–11488. [PubMed] [Google Scholar]
  25. Karczewski P., Bartel S., Haase H., Krause E. G. Isoproterenol induces both cAMP- and calcium-dependent phosphorylation of phospholamban in canine heart in vivo. Biomed Biochim Acta. 1987;46(8-9):S433–S439. [PubMed] [Google Scholar]
  26. Karim C. B., Stamm J. D., Karim J., Jones L. R., Thomas D. D. Cysteine reactivity and oligomeric structures of phospholamban and its mutants. Biochemistry. 1998 Sep 1;37(35):12074–12081. doi: 10.1021/bi980642n. [DOI] [PubMed] [Google Scholar]
  27. Katz A. M., Tada M., Kirchberger M. A. Control of calcium transport in the myocardium by the cyclic AMP-Protein kinase system. Adv Cyclic Nucleotide Res. 1975;5:453–472. [PubMed] [Google Scholar]
  28. Kimura Y., Kurzydlowski K., Tada M., MacLennan D. H. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem. 1997 Jun 13;272(24):15061–15064. doi: 10.1074/jbc.272.24.15061. [DOI] [PubMed] [Google Scholar]
  29. Kimura Y., Kurzydlowski K., Tada M., MacLennan D. H. Phospholamban regulates the Ca2+-ATPase through intramembrane interactions. J Biol Chem. 1996 Sep 6;271(36):21726–21731. doi: 10.1074/jbc.271.36.21726. [DOI] [PubMed] [Google Scholar]
  30. Kirchberber M. A., Tada M., Katz A. M. Phospholamban: a regulatory protein of the cardiac sarcoplasmic reticulum. Recent Adv Stud Cardiac Struct Metab. 1975;5:103–115. [PubMed] [Google Scholar]
  31. Kleinschmidt J. H., Mahaney J. E., Thomas D. D., Marsh D. Interaction of bee venom melittin with zwitterionic and negatively charged phospholipid bilayers: a spin-label electron spin resonance study. Biophys J. 1997 Feb;72(2 Pt 1):767–778. doi: 10.1016/s0006-3495(97)78711-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kovacs R. J., Nelson M. T., Simmerman H. K., Jones L. R. Phospholamban forms Ca2+-selective channels in lipid bilayers. J Biol Chem. 1988 Dec 5;263(34):18364–18368. [PubMed] [Google Scholar]
  33. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  34. Laskowski R. A., Rullmannn J. A., MacArthur M. W., Kaptein R., Thornton J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR. 1996 Dec;8(4):477–486. doi: 10.1007/BF00228148. [DOI] [PubMed] [Google Scholar]
  35. Ludlam C. F., Arkin I. T., Liu X. M., Rothman M. S., Rath P., Aimoto S., Smith S. O., Engelman D. M., Rothschild K. J. Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban. Biophys J. 1996 Apr;70(4):1728–1736. doi: 10.1016/S0006-3495(96)79735-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Malashkevich V. N., Kammerer R. A., Efimov V. P., Schulthess T., Engel J. The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel? Science. 1996 Nov 1;274(5288):761–765. doi: 10.1126/science.274.5288.761. [DOI] [PubMed] [Google Scholar]
  37. Maslennikov I. V., Sobol A. G., Anagli J., James P., Vorherr T., Arseniev A. S., Carafoli E. The secondary structure of phospholamban: a two-dimensional NMR study. Biochem Biophys Res Commun. 1995 Dec 26;217(3):1200–1207. doi: 10.1006/bbrc.1995.2896. [DOI] [PubMed] [Google Scholar]
  38. Meyer M., Schillinger W., Pieske B., Holubarsch C., Heilmann C., Posival H., Kuwajima G., Mikoshiba K., Just H., Hasenfuss G. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation. 1995 Aug 15;92(4):778–784. doi: 10.1161/01.cir.92.4.778. [DOI] [PubMed] [Google Scholar]
  39. Mortishire-Smith R. J., Pitzenberger S. M., Burke C. J., Middaugh C. R., Garsky V. M., Johnson R. G. Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure. Biochemistry. 1995 Jun 13;34(23):7603–7613. doi: 10.1021/bi00023a006. [DOI] [PubMed] [Google Scholar]
  40. Quirk P. G., Patchell V. B., Colyer J., Drago G. A., Gao Y. Conformational effects of serine phosphorylation in phospholamban peptides. Eur J Biochem. 1996 Feb 15;236(1):85–91. doi: 10.1111/j.1432-1033.1996.00085.x. [DOI] [PubMed] [Google Scholar]
  41. Reddy L. G., Jones L. R., Cala S. E., O'Brian J. J., Tatulian S. A., Stokes D. L. Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca(2+)-ATPase. J Biol Chem. 1995 Apr 21;270(16):9390–9397. doi: 10.1074/jbc.270.16.9390. [DOI] [PubMed] [Google Scholar]
  42. Rothemund S., Weisshoff H., Beyermann M., Krause E., Bienert M., Mügge C., Sykes B. D., Sönnichsen F. D. Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: a monitor of intramolecular hydrogen bonds in helical peptides. J Biomol NMR. 1996 Jul;8(1):93–97. doi: 10.1007/BF00198143. [DOI] [PubMed] [Google Scholar]
  43. Sasaki T., Inui M., Kimura Y., Kuzuya T., Tada M. Molecular mechanism of regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum. Effects of synthetic phospholamban peptides on Ca2+ pump ATPase. J Biol Chem. 1992 Jan 25;267(3):1674–1679. [PubMed] [Google Scholar]
  44. Simmerman H. K., Collins J. H., Theibert J. L., Wegener A. D., Jones L. R. Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains. J Biol Chem. 1986 Oct 5;261(28):13333–13341. [PubMed] [Google Scholar]
  45. Simmerman H. K., Kobayashi Y. M., Autry J. M., Jones L. R. A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J Biol Chem. 1996 Mar 8;271(10):5941–5946. doi: 10.1074/jbc.271.10.5941. [DOI] [PubMed] [Google Scholar]
  46. Simmerman H. K., Lovelace D. E., Jones L. R. Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum. Biochim Biophys Acta. 1989 Aug 31;997(3):322–329. doi: 10.1016/0167-4838(89)90203-3. [DOI] [PubMed] [Google Scholar]
  47. Starling A. P., Sharma R. P., East J. M., Lee A. G. The effect of N-terminal acetylation on Ca(2+)-ATPase inhibition by phospholamban. Biochem Biophys Res Commun. 1996 Sep 13;226(2):352–355. doi: 10.1006/bbrc.1996.1360. [DOI] [PubMed] [Google Scholar]
  48. Sönnichsen F. D., Van Eyk J. E., Hodges R. S., Sykes B. D. Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide. Biochemistry. 1992 Sep 22;31(37):8790–8798. doi: 10.1021/bi00152a015. [DOI] [PubMed] [Google Scholar]
  49. Tada M., Kadoma M. Regulation of the Ca2+ pump ATPase by cAMP-dependent phosphorylation of phospholamban. Bioessays. 1989 May;10(5):157–163. doi: 10.1002/bies.950100505. [DOI] [PubMed] [Google Scholar]
  50. Tatulian S. A., Jones L. R., Reddy L. G., Stokes D. L., Tamm L. K. Secondary structure and orientation of phospholamban reconstituted in supported bilayers from polarized attenuated total reflection FTIR spectroscopy. Biochemistry. 1995 Apr 4;34(13):4448–4456. doi: 10.1021/bi00013a038. [DOI] [PubMed] [Google Scholar]
  51. Terzi E., Poteur L., Trifilieff E. Evidence for a phosphorylation-induced conformational change in phospholamban cytoplasmic domain by CD analysis. FEBS Lett. 1992 Sep 14;309(3):413–416. doi: 10.1016/0014-5793(92)80819-3. [DOI] [PubMed] [Google Scholar]
  52. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum. J Biol Chem. 1994 Jan 28;269(4):3088–3094. [PubMed] [Google Scholar]
  53. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban. J Biol Chem. 1994 Sep 16;269(37):22929–22932. [PubMed] [Google Scholar]
  54. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban. J Biol Chem. 1993 Feb 5;268(4):2809–2815. [PubMed] [Google Scholar]
  55. Vorherr T., Chiesi M., Schwaller R., Carafoli E. Regulation of the calcium ion pump of sarcoplasmic reticulum: reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump. Biochemistry. 1992 Jan 21;31(2):371–376. doi: 10.1021/bi00117a009. [DOI] [PubMed] [Google Scholar]
  56. Vorherr T., Wrzosek A., Chiesi M., Carafoli E. Total synthesis and functional properties of the membrane-intrinsic protein phospholamban. Protein Sci. 1993 Mar;2(3):339–347. doi: 10.1002/pro.5560020306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Voss J. C., Mahaney J. E., Thomas D. D. Mechanism of Ca-ATPase inhibition by melittin in skeletal sarcoplasmic reticulum. Biochemistry. 1995 Jan 24;34(3):930–939. doi: 10.1021/bi00003a027. [DOI] [PubMed] [Google Scholar]
  58. Wishart D. S., Sykes B. D., Richards F. M. Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy. FEBS Lett. 1991 Nov 18;293(1-2):72–80. doi: 10.1016/0014-5793(91)81155-2. [DOI] [PubMed] [Google Scholar]
  59. Wishart D. S., Sykes B. D., Richards F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992 Feb 18;31(6):1647–1651. doi: 10.1021/bi00121a010. [DOI] [PubMed] [Google Scholar]
  60. Wüthrich K., Billeter M., Braun W. Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance. J Mol Biol. 1983 Oct 5;169(4):949–961. doi: 10.1016/s0022-2836(83)80144-2. [DOI] [PubMed] [Google Scholar]
  61. Yeagle P. L., Alderfer J. L., Albert A. D. Three-dimensional structure of the cytoplasmic face of the G protein receptor rhodopsin. Biochemistry. 1997 Aug 12;36(32):9649–9654. doi: 10.1021/bi970908a. [DOI] [PubMed] [Google Scholar]

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