Advertisement

Journal of Muscle Research & Cell Motility

, Volume 21, Issue 6, pp 537–549 | Cite as

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

  • Giancarlo Notarianni
  • Nikolai Gusev
  • Daniel Lafitte
  • Tessa J. Hill
  • Helen S. Cooper
  • Peter J. Derrick
  • Steven B. Marston
Article

Abstract

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25°C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.

Keywords

DEAE Smooth Muscle Actin Thin Filament Aqueous Buffer Tryptophan Fluorescence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bayley PM, Findlay WA and Martin SR (1996) Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences. Protein Sci 5: 1215–1228.Google Scholar
  2. Chacko S and Eisenberg E (1990) Cooperativity of actin-activated ATPase of gizzard heavy meromyosin in the presence of gizzard tropomyosin. J Biol Chem 265: 2105–2110.Google Scholar
  3. Crivici A and Ikura M (1995) Molecular and structural basis of target recognition by calmodulin Annu Rev Biophys Biomol Struct 24: 85–116.Google Scholar
  4. Dabrowska R, Goch A, Galazkiewicz B and Osinska H (1985) The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta 842: 70–75.Google Scholar
  5. Fraser IDC, Copeland O, Wu B and Marston SB (1997) The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4. Biochemistry 36: 5483–5492.Google Scholar
  6. Fraser IDC and Marston SB (1995) In vitro motility analysis of smooth muscle caldesmon control of actin-tropomyosin filament movement. J Biol Chem 270: 19688–19693.Google Scholar
  7. Fujii T, Machino K, Andoh H, Satoh T and Kondo Y (1990) Calcium-dependent control of caldesmon-actin interaction by S100 protein. J Biochem (Tokyo) 107: 133–137.Google Scholar
  8. Gao Y, Patchell VB, Huber PAJ, Copeland O, EL-Mezgueldi M, Fattoum A, Calas B, Thorsted P, Marston S and Levine B (1999) The interface between caldesmon domain 4b and subdomain 1 of actin studied by nuclear magnetic resonance spectroscopy. Biochemistry 38: 15459–15469.Google Scholar
  9. Gopalakrishna R and Anderson WB (1982) Ca2+ induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-sepharose affinity chromatography. Biochem Biophys Res Commun 104: 830–836.Google Scholar
  10. Gopalakrishna R and Anderson WB (1985) Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins. J Appl Biochem 7: 311–322.Google Scholar
  11. Graceffa P (1999) Movement of smooth muscle tropomyosin by myosin heads. Biochemistry 38: 11984–11992.Google Scholar
  12. Huber PAJ, EL-Mezgueldi M, Grabarek Z, Slatter DA, Levine BA and Marston SB (1996) Multiple sited interaction of caldesmon with Ca2+.calmodulin. Biochem J 316: 413–420.Google Scholar
  13. Humphrey MB, Herrera-Sosa H, Gonzalez G, Lee R and Bryan J (1992) Molecular cloning of human caldesmons. Gene 112: 197–205.Google Scholar
  14. Ikura G, Clore GM, Gronenborn AM, Zhu G, Klee CB and Bax A (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256: 632–638.Google Scholar
  15. Kakiuchi R, Inui M, Morimoto K, Kanda K, Sobue K and Kakiuchi S (1983) Çaldesmon, a calmodulin binding, F-actin interacting protein, is present in aorta, uterus and platelets. FEBS Lett 154: 351–356.Google Scholar
  16. Krueger JK, Galllagher SC, Wang C-LA and Trewella J (2000) Calmodulin remains extended upon binding to smooth muscle caldesmon: a combined small-angle scattering and Fourier trans-form infrared spectroscopy study. Biochemistry 39: 3979–3987.Google Scholar
  17. Lafitte D, Capony JP, Grassy G, Haiech J and Calas B (1995) Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry. Biochemistry 34: 13825–13832.Google Scholar
  18. Lafitte D, Heck AJ, Hill TJ, Jumel K, Harding SE and Derrick PJ (1999) Evidence of noncovalent dimerization of calmodulin. Eur J Biochem 261: 337–344.Google Scholar
  19. Lavanant H, Derrick PJ, Heck AJ and Mellon FA (1998) Analysis of nisin A and some of its variants using Fourier transform ion cyclotron resonance mass spectrometry. Anal Biochem 255: 74–89.Google Scholar
  20. Lehrer SS and Geeves MA (1998) The muscle thin filament as a classical cooperative/allosteric regulatory system. J Mol Biol 277: 1081–1089.Google Scholar
  21. Lehrer SS, Golitsina NL and Geeves MA (1998) Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions. Biochemistry 36: 13449–13454.Google Scholar
  22. Mabuchi Y, Wang C-LA and Grabarek Z (1995) Calmodulin has an extended conformation when complexed with smooth muscle caldesmon. Biophys J 68: A359.Google Scholar
  23. Mani RS, McCubbin WD and Kay CM (1992) Calcium-dependent regulation of caldesmon by an 11 kDa smooth muscle calcium binding protein, caltropin. Biochemistry 31: 11896–11901.Google Scholar
  24. Margossian SS and Lowey S (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 85: 55–71.Google Scholar
  25. Marston S, Burton D, Copeland O, Fraser I, Gao Y, Hodgkinson J, Huber P, Levine B, EL-Mezgueldi M and Notarianni G (1998) Structural interactions between actin, tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments. Acta Physiol Scand 164: 401–414.Google Scholar
  26. Marston SB (1982) The regulation of smooth muscle contractile proteins. Prog Biophys Molec Biol 41: 1–41.Google Scholar
  27. Marston SB (1990) Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle. Biochem J 272: 305–310.Google Scholar
  28. Marston SB, Fraser IDC and Huber PAJ (1994) Smooth muscle caldesmon controls the strong binding interactions between actin-tropomyosin and myosin. J Biol Chem 269: 32104–32109.Google Scholar
  29. Marston SB and Huber PAJ (1996) Caldesmon. In: Barany M and Barany K (eds.) Biochemistry of Smooth Muscle Contraction, (pp. 77–90). Academic Press, San Diego.Google Scholar
  30. Marston SB and Smith CWJ (1984) Purification and properties of Ca2+-regulated thin filaments and f-actin from sheep aorta smooth muscle. J Muscle Res Cell Motil 5: 559–575.Google Scholar
  31. Marston SB, Trevett RM and Walters M (1980) Calcium ion regulated thin filaments from vascular smooth muscle. Biochem J 185: 355–365.Google Scholar
  32. Medvedeva MV, Bushueva TL, Shirinsky VP, Lukas TJ, Watterson DM and Gusev NB (1995) Interaction of smooth muscle caldesmon with calmodulin mutants. FEBS Lett 360: 89–92.Google Scholar
  33. Medvedeva MV, Kolobova EA, Huber PAJ, Fraser IDC, Marston SB and Gusev NB (1997) Mapping of contact sites in the caldesmon-calmodulin complex. Biochem J 324: 255–262.Google Scholar
  34. Meyer DF, Mabuchi Y and Grabarek Z (1996) The role of Phe92 in the Ca2+-induced conformational transition in the C-terminal domain of calmodulin. J Biol Chem 271: 11284–11290.Google Scholar
  35. Newton DL, Oldewurtel MD, Krinks MH, Shiloach J and Klee CB (1983) Agonist and antagonist properties of calmodulin fragments. J Biol Chem 259: 4419–4426.Google Scholar
  36. Ngai PK and Walsh MP (1984) Inhibition of smooth muscle actin-activated Myosin Mg2+ ATPase activity by caldesmon. J Biol Chem 259: 13656–13659.Google Scholar
  37. Polyakov AA and Gusev NB (1997) Utilization of troponin C as a model calcium-binding protein for mapping the calmodulin binding sites of caldesmon. Biochem J 321: 873–878.Google Scholar
  38. Polyakov AA, Huber PAJ, Marston SB and Gusev NB (1998) Interaction of isoforms of S100 protein with smooth muscle caldesmon. FEBS Lett 422: 235–239.Google Scholar
  39. Pritchard K and Marston SB (1988) The control of calcium-regulated thin filaments from vascular smooth muscle by calmodulin and other calcium binding proteins. Biochem Soc Trans 16: 355–356.Google Scholar
  40. Pritchard K and Marston SB (1989) Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments. Biochem J 257: 839–843.Google Scholar
  41. Pritchard K and Marston SB (1991) Ca2+ dependent regulation of vascular smooth muscle caldesmon by S.100 and related smooth muscle proteins. Biochem J 277: 819–824.Google Scholar
  42. Pritchard KP and Marston SB (1993) The Ca2+ sensitising compo-nents of smooth muscle thin filaments: properties of regulatory factors that interact with caldesmon. Biochem Biophys Res Comm 190: 668–673.Google Scholar
  43. Sheterline P, Clayton P and Sparrow JC (1998) Actin. Oxford University Press, Oxford.Google Scholar
  44. Shirinsky VP, Bushueva TL and Frolova SI (1988) Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence. Biochem J 255: 203–208.Google Scholar
  45. Skripnikova EV and Gusev NB (1989) Interaction of smooth muscle caldesmon with S-100 protein. FEBS Lett 257: 380–382.Google Scholar
  46. Smith CW, Pritchard K and Marston SB (1987) The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin. J Biol Chem 262: 116–122.Google Scholar
  47. Smith CWJ and Marston SB (1985) Disassembly and reconstitution of the Ca2+-sensitive thin filaments of vascular smooth muscle. FEBS Lett 184: 115–119.Google Scholar
  48. Sobue K, Morimoto K, Inui M, Kanda K and Kakiuchi S (1982) Control of actin-myosin interaction of gizzard smooth muscle by calmodulin and caldesmon-linked flip-flop mechanism. Biomed Res 3: 188–196.Google Scholar
  49. Sobue K, Muramoto Y, Fujita M and Kakiuchi S (1981) Purification of a calmodulin binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci USA 78: 5652–5655.Google Scholar
  50. Straub FB (1942) Actin. Studies from the Institute of Medical Chemistry, University of Szeged 2: 3–16.Google Scholar
  51. Taussky HH and Schorr E (1953) A microscopic method for the determination of inorganic phosphorus. J Biol Chem 202: 675–685.Google Scholar
  52. Tsvetkov PO, Protasevich II, Gilli R, Lafitte D, Lobachov VM, Haiech J, Briand C and Makarov AA (1999) Apocalmodulin binds to the myosin light chain kinase calmodulin target site. J Biol Chem 274: 18161–18164.Google Scholar
  53. West CA, Bannister JV, Levine BA and Perham RN (1988) Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein. Protein Eng 2: 307–311.Google Scholar
  54. Yuan T, Walsh MP, Sutherland C, Fabian H and Vogel HJ (1999) Calcium-dependent and-independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin. Biochemistry 38: 1446–1455.Google Scholar
  55. Zhou N, Yuan T, Mak AS and Vogel HJ (1997) NMR studies of caldesmon-calmodulin interactions. Biochemistry 36: 2817–2825.Google Scholar
  56. Zhuang S, Mani RS, Kay CM and Wang C-LA (1995) Interaction between caltropin and the C-terminal region of smooth muscle caldesmon. Biochem Biophys Res Commun 209: 12–17.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Giancarlo Notarianni
    • 1
  • Nikolai Gusev
    • 2
  • Daniel Lafitte
    • 3
  • Tessa J. Hill
    • 3
  • Helen S. Cooper
    • 3
  • Peter J. Derrick
    • 3
  • Steven B. Marston
    • 1
  1. 1.Imperial College School of MedicineNational Heart and Lung InstituteLondonUK
  2. 2.Department of Biochemistry, School of BiologyMoscow State UniversityMoscowRussia
  3. 3.Institute of Mass Spectrometry and Department of ChemistryUniversity of WarwickCoventryUK

Personalised recommendations