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. 1996 Oct;71(4):1920–1933. doi: 10.1016/S0006-3495(96)79391-8

Affinity and structure of complexes of tropomyosin and caldesmon domains.

E J Hnath 1, C L Wang 1, P A Huber 1, S B Marston 1, G N Phillips Jr 1
PMCID: PMC1233659  PMID: 8889167

Abstract

The interaction of caldesmon domains with tropomyosin has been studied using x-ray crystallography and an optical biosensor. Only whole caldesmon and the carboxyl-terminal domain of caldesmon (CaD-4, chicken gizzard residues 597-756) bound to tropomyosin with greater than millimolar affinity at 100 and 150 microM salt. Under these conditions the affinities of whole caldesmon and CaD-4 were both in the micromolar range. Data from the x-ray studies showed that whole caldesmon bound to tropomyosin in several places, with the region of tightest interaction being at tropomyosin residues 70-100 and/or 230-260. Studies with CaD-4 revealed that this region corresponded to the strong binding site seen with whole caldesmon. Weaker association of other regions of caldesmon to tropomyosin residues 180-210 and 5-50 was also observed. The results suggest that the carboxyl-terminus of caldesmon binds tightly to tropomyosin and that other regions of caldesmon may interact with tropomyosin tightly only when they are held close to tropomyosin by the carboxyl-terminal domain. Four models are presented to show the possible interactions of caldesmon with tropomyosin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen B. G., Walsh M. P. The biochemical basis of the regulation of smooth-muscle contraction. Trends Biochem Sci. 1994 Sep;19(9):362–368. doi: 10.1016/0968-0004(94)90112-0. [DOI] [PubMed] [Google Scholar]
  2. Bailey K. Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem J. 1948;43(2):271–279. doi: 10.1042/bj0430271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartegi A., Fattoum A., Derancourt J., Kassab R. Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region. J Biol Chem. 1990 Sep 5;265(25):15231–15238. [PubMed] [Google Scholar]
  4. Bryan J., Imai M., Lee R., Moore P., Cook R. G., Lin W. G. Cloning and expression of a smooth muscle caldesmon. J Biol Chem. 1989 Aug 15;264(23):13873–13879. [PubMed] [Google Scholar]
  5. Chacko S., Phillips G. N., Jr Diffuse x-ray scattering from tropomyosin crystals. Biophys J. 1992 May;61(5):1256–1266. doi: 10.1016/S0006-3495(92)81934-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dabrowska R., Goch A., Gałazkiewicz B., Osińska H. The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta. 1985 Sep 27;842(1):70–75. doi: 10.1016/0304-4165(85)90295-8. [DOI] [PubMed] [Google Scholar]
  7. Dedman J. R., Kaetzel M. A. Calmodulin purification and fluorescent labeling. Methods Enzymol. 1983;102:1–8. doi: 10.1016/s0076-6879(83)02003-0. [DOI] [PubMed] [Google Scholar]
  8. Edwards P. R., Gill A., Pollard-Knight D. V., Hoare M., Buckle P. E., Lowe P. A., Leatherbarrow R. J. Kinetics of protein-protein interactions at the surface of an optical biosensor. Anal Biochem. 1995 Oct 10;231(1):210–217. doi: 10.1006/abio.1995.1522. [DOI] [PubMed] [Google Scholar]
  9. Fraser I. D., Marston S. B. In vitro motility analysis of smooth muscle caldesmon control of actin-tropomyosin filament movement. J Biol Chem. 1995 Aug 25;270(34):19688–19693. doi: 10.1074/jbc.270.34.19688. [DOI] [PubMed] [Google Scholar]
  10. George A. J., French R. R., Glennie M. J. Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor. J Immunol Methods. 1995 Jun 14;183(1):51–63. doi: 10.1016/0022-1759(95)00031-5. [DOI] [PubMed] [Google Scholar]
  11. Graceffa P. Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin. J Biol Chem. 1995 Dec 15;270(50):30187–30193. [PubMed] [Google Scholar]
  12. Hegmann T. E., Schulte D. L., Lin J. L., Lin J. J. Inhibition of intracellular granule movement by microinjection of monoclonal antibodies against caldesmon. Cell Motil Cytoskeleton. 1991;20(2):109–120. doi: 10.1002/cm.970200204. [DOI] [PubMed] [Google Scholar]
  13. Hemric M. E., Freedman M. V., Chalovich J. M. Inhibition of actin stimulation of skeletal muscle (A1)S-1 ATPase activity by caldesmon. Arch Biochem Biophys. 1993 Oct;306(1):39–43. doi: 10.1006/abbi.1993.1477. [DOI] [PubMed] [Google Scholar]
  14. Hitchcock-DeGregori S. E., Heald R. W. Altered actin and troponin binding of amino-terminal variants of chicken striated muscle alpha-tropomyosin expressed in Escherichia coli. J Biol Chem. 1987 Jul 15;262(20):9730–9735. [PubMed] [Google Scholar]
  15. Huber P. A., Fraser I. D., Marston S. B. Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon. Biochem J. 1995 Dec 1;312(Pt 2):617–625. doi: 10.1042/bj3120617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katayama E., Ikebe M. Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament. Biophys J. 1995 Jun;68(6):2419–2428. doi: 10.1016/S0006-3495(95)80424-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katayama E., Scott-Woo G., Ikebe M. Effect of caldesmon on the assembly of smooth muscle myosin. J Biol Chem. 1995 Feb 24;270(8):3919–3925. doi: 10.1074/jbc.270.8.3919. [DOI] [PubMed] [Google Scholar]
  18. Katsuyama H., Wang C. L., Morgan K. G. Regulation of vascular smooth muscle tone by caldesmon. J Biol Chem. 1992 Jul 25;267(21):14555–14558. [PubMed] [Google Scholar]
  19. Kraft T., Chalovich J. M., Yu L. C., Brenner B. Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation. Biophys J. 1995 Jun;68(6):2404–2418. doi: 10.1016/S0006-3495(95)80423-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Lamb N. J., Fernandez A., Mezgueldi M., Labbé J. P., Kassab R., Fattoum A. Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon. Eur J Cell Biol. 1996 Jan;69(1):36–44. [PubMed] [Google Scholar]
  22. Lynch W., Bretscher A. Purification of caldesmon. Methods Enzymol. 1986;134:37–42. doi: 10.1016/0076-6879(86)34073-4. [DOI] [PubMed] [Google Scholar]
  23. Mabuchi K., Lin J. J., Wang C. L. Electron microscopic images suggest both ends of caldesmon interact with actin filaments. J Muscle Res Cell Motil. 1993 Feb;14(1):54–64. doi: 10.1007/BF00132180. [DOI] [PubMed] [Google Scholar]
  24. Mabuchi K., Wang C. L. Electron microscopic studies of chicken gizzard caldesmon and its complex with calmodulin. J Muscle Res Cell Motil. 1991 Apr;12(2):145–151. doi: 10.1007/BF01774033. [DOI] [PubMed] [Google Scholar]
  25. Marston S. B., Fraser I. D., Huber P. A., Pritchard K., Gusev N. B., Torok K. Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin. J Biol Chem. 1994 Mar 18;269(11):8134–8139. [PubMed] [Google Scholar]
  26. Marston S. B., Fraser I. D., Huber P. A. Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin. J Biol Chem. 1994 Dec 23;269(51):32104–32109. [PubMed] [Google Scholar]
  27. Marston S. B., Redwood C. S., Lehman W. Reversal of caldesmon function by anti-caldesmon antibodies confirms its role in the calcium regulation of vascular smooth muscle thin filaments. Biochem Biophys Res Commun. 1988 Aug 30;155(1):197–202. doi: 10.1016/s0006-291x(88)81068-4. [DOI] [PubMed] [Google Scholar]
  28. Marston S. B., Redwood C. S. The molecular anatomy of caldesmon. Biochem J. 1991 Oct 1;279(Pt 1):1–16. doi: 10.1042/bj2790001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marston S. B., Smith C. W. The thin filaments of smooth muscles. J Muscle Res Cell Motil. 1985 Dec;6(6):669–708. doi: 10.1007/BF00712237. [DOI] [PubMed] [Google Scholar]
  30. Mezgueldi M., Derancourt J., Calas B., Kassab R., Fattoum A. Precise identification of the regulatory F-actin- and calmodulin-binding sequences in the 10-kDa carboxyl-terminal domain of caldesmon. J Biol Chem. 1994 Apr 29;269(17):12824–12832. [PubMed] [Google Scholar]
  31. Moody C., Lehman W., Craig R. Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments. J Muscle Res Cell Motil. 1990 Apr;11(2):176–185. doi: 10.1007/BF01766496. [DOI] [PubMed] [Google Scholar]
  32. Morton T. A., Myszka D. G., Chaiken I. M. Interpreting complex binding kinetics from optical biosensors: a comparison of analysis by linearization, the integrated rate equation, and numerical integration. Anal Biochem. 1995 May 1;227(1):176–185. doi: 10.1006/abio.1995.1268. [DOI] [PubMed] [Google Scholar]
  33. Pearlstone J. R., Smillie L. B. Binding of troponin-T fragments to several types of tropomyosin. Sensitivity to Ca2+ in the presence of troponin-C. J Biol Chem. 1982 Sep 25;257(18):10587–10592. [PubMed] [Google Scholar]
  34. Pfitzer G., Zeugner C., Troschka M., Chalovich J. M. Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5904–5908. doi: 10.1073/pnas.90.13.5904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Phillips G. N., Jr Diffraction methods for biological macromolecules. Crystallization in capillary tubes. Methods Enzymol. 1985;114:128–131. doi: 10.1016/0076-6879(85)14010-3. [DOI] [PubMed] [Google Scholar]
  36. Redwood C. S., Marston S. B. Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon. J Biol Chem. 1993 May 25;268(15):10969–10976. [PubMed] [Google Scholar]
  37. Smith C. W., Pritchard K., Marston S. B. The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin. J Biol Chem. 1987 Jan 5;262(1):116–122. [PubMed] [Google Scholar]
  38. Sobue K., Muramoto Y., Fujita M., Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. doi: 10.1073/pnas.78.9.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Szabo A., Stolz L., Granzow R. Surface plasmon resonance and its use in biomolecular interaction analysis (BIA). Curr Opin Struct Biol. 1995 Oct;5(5):699–705. doi: 10.1016/0959-440x(95)80064-6. [DOI] [PubMed] [Google Scholar]
  40. Taggart M. J., Marston S. B. The effects of vascular smooth muscle caldesmon on force production by 'desensitised' skeletal muscle fibres. FEBS Lett. 1988 Dec 19;242(1):171–174. doi: 10.1016/0014-5793(88)81009-3. [DOI] [PubMed] [Google Scholar]
  41. Tsuruda T. S., Watson M. H., Foster D. B., Lin J. J., Mak A. S. Alignment of caldesmon on the actin-tropomyosin filaments. Biochem J. 1995 Aug 1;309(Pt 3):951–957. doi: 10.1042/bj3090951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vibert P., Craig R., Lehman W. Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments. J Cell Biol. 1993 Oct;123(2):313–321. doi: 10.1083/jcb.123.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang C. L., Wang L. W., Xu S. A., Lu R. C., Saavedra-Alanis V., Bryan J. Localization of the calmodulin- and the actin-binding sites of caldesmon. J Biol Chem. 1991 May 15;266(14):9166–9172. [PubMed] [Google Scholar]
  44. Wang Z., Horiuchi K. Y., Chacko S. Characterization of the functional domains on the C-terminal region of caldesmon using full-length and mutant caldesmon molecules. J Biol Chem. 1996 Jan 26;271(4):2234–2242. doi: 10.1074/jbc.271.4.2234. [DOI] [PubMed] [Google Scholar]
  45. Warren K. S., Lin J. L., Wamboldt D. D., Lin J. J. Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments. J Cell Biol. 1994 Apr;125(2):359–368. doi: 10.1083/jcb.125.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Watson M. H., Kuhn A. E., Mak A. S. Caldesmon, calmodulin and tropomyosin interactions. Biochim Biophys Acta. 1990 Aug 13;1054(1):103–113. doi: 10.1016/0167-4889(90)90211-u. [DOI] [PubMed] [Google Scholar]
  47. Watson M. H., Kuhn A. E., Novy R. E., Lin J. J., Mak A. S. Caldesmon-binding sites on tropomyosin. J Biol Chem. 1990 Nov 5;265(31):18860–18866. [PubMed] [Google Scholar]
  48. Whitby F. G., Kent H., Stewart F., Stewart M., Xie X., Hatch V., Cohen C., Phillips G. N., Jr Structure of tropomyosin at 9 angstroms resolution. J Mol Biol. 1992 Sep 20;227(2):441–452. doi: 10.1016/0022-2836(92)90899-u. [DOI] [PubMed] [Google Scholar]
  49. White S. P., Cohen C., Phillips G. N., Jr Structure of co-crystals of tropomyosin and troponin. 1987 Feb 26-Mar 4Nature. 325(6107):826–828. doi: 10.1038/325826a0. [DOI] [PubMed] [Google Scholar]
  50. Yamashiro-Matsumura S., Matsumura F. Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin. J Cell Biol. 1988 Jun;106(6):1973–1983. doi: 10.1083/jcb.106.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhuang S., Wang E., Wang C. L. Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon. J Biol Chem. 1995 Aug 25;270(34):19964–19968. doi: 10.1074/jbc.270.34.19964. [DOI] [PubMed] [Google Scholar]

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