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. 2001 Jul;81(1):321–333. doi: 10.1016/S0006-3495(01)75702-5

Proximity relationships between residue 117 of rabbit skeletal troponin-I and residues in troponin-C and actin.

Z Li 1, J Gergely 1, T Tao 1
PMCID: PMC1301514  PMID: 11423417

Abstract

We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.

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Selected References

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  1. Dalbey R. E., Weiel J., Yount R. G. Förster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1. Biochemistry. 1983 Sep 27;22(20):4696–4706. doi: 10.1021/bi00289a014. [DOI] [PubMed] [Google Scholar]
  2. Fairclough R. H., Cantor C. R. The use of singlet-singlet energy transfer to study macromolecular assemblies. Methods Enzymol. 1978;48:347–379. doi: 10.1016/s0076-6879(78)48019-x. [DOI] [PubMed] [Google Scholar]
  3. Farah C. S., Miyamoto C. A., Ramos C. H., da Silva A. C., Quaggio R. B., Fujimori K., Smillie L. B., Reinach F. C. Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I. J Biol Chem. 1994 Feb 18;269(7):5230–5240. [PubMed] [Google Scholar]
  4. Farah C. S., Reinach F. C. The troponin complex and regulation of muscle contraction. FASEB J. 1995 Jun;9(9):755–767. doi: 10.1096/fasebj.9.9.7601340. [DOI] [PubMed] [Google Scholar]
  5. Fujimori K., Sorenson M., Herzberg O., Moult J., Reinach F. C. Probing the calcium-induced conformational transition of troponin C with site-directed mutants. Nature. 1990 May 10;345(6271):182–184. doi: 10.1038/345182a0. [DOI] [PubMed] [Google Scholar]
  6. Gagné S. M., Tsuda S., Li M. X., Smillie L. B., Sykes B. D. Structures of the troponin C regulatory domains in the apo and calcium-saturated states. Nat Struct Biol. 1995 Sep;2(9):784–789. doi: 10.1038/nsb0995-784. [DOI] [PubMed] [Google Scholar]
  7. Gong B. J., Mabuchi K., Takahashi K., Nadal-Ginard B., Tao T. Characterization of wild type and mutant chicken gizzard alpha calponin expressed in E. coli. J Biochem. 1993 Oct;114(4):453–456. doi: 10.1093/oxfordjournals.jbchem.a124197. [DOI] [PubMed] [Google Scholar]
  8. Gordon A. M., Homsher E., Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000 Apr;80(2):853–924. doi: 10.1152/physrev.2000.80.2.853. [DOI] [PubMed] [Google Scholar]
  9. Grabarek Z., Drabikowski W., Leavis P. C., Rosenfeld S. S., Gergely J. Proteolytic fragments of troponin C. Interactions with the other troponin subunits and biological activity. J Biol Chem. 1981 Dec 25;256(24):13121–13127. [PubMed] [Google Scholar]
  10. Grabarek Z., Tan R. Y., Wang J., Tao T., Gergely J. Inhibition of mutant troponin C activity by an intra-domain disulphide bond. Nature. 1990 May 10;345(6271):132–135. doi: 10.1038/345132a0. [DOI] [PubMed] [Google Scholar]
  11. Grabarek Z., Tao T., Gergely J. Molecular mechanism of troponin-C function. J Muscle Res Cell Motil. 1992 Aug;13(4):383–393. doi: 10.1007/BF01738034. [DOI] [PubMed] [Google Scholar]
  12. Greaser M. L., Gergely J. Purification and properties of the components from troponin. J Biol Chem. 1973 Mar 25;248(6):2125–2133. [PubMed] [Google Scholar]
  13. Greaser M. L., Gergely J. Reconstitution of troponin activity from three protein components. J Biol Chem. 1971 Jul 10;246(13):4226–4233. [PubMed] [Google Scholar]
  14. Herzberg O., James M. N. Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nature. 1985 Feb 21;313(6004):653–659. doi: 10.1038/313653a0. [DOI] [PubMed] [Google Scholar]
  15. Herzberg O., Moult J., James M. N. A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction. J Biol Chem. 1986 Feb 25;261(6):2638–2644. [PubMed] [Google Scholar]
  16. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  17. Houdusse A., Love M. L., Dominguez R., Grabarek Z., Cohen C. Structures of four Ca2+-bound troponin C at 2.0 A resolution: further insights into the Ca2+-switch in the calmodulin superfamily. Structure. 1997 Dec 15;5(12):1695–1711. doi: 10.1016/s0969-2126(97)00315-8. [DOI] [PubMed] [Google Scholar]
  18. Jha P. K., Mao C., Sarkar S. Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: the inhibitory region enhances binding of troponin I fragments to troponin C. Biochemistry. 1996 Aug 27;35(34):11026–11035. doi: 10.1021/bi960406h. [DOI] [PubMed] [Google Scholar]
  19. Kobayashi T., Kobayashi M., Gryczynski Z., Lakowicz J. R., Collins J. H. Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments. Biochemistry. 2000 Jan 11;39(1):86–91. doi: 10.1021/bi991903b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kobayashi T., Tao T., Gergely J., Collins J. H. Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants. J Biol Chem. 1994 Feb 25;269(8):5725–5729. [PubMed] [Google Scholar]
  21. Kobayashi T., Tao T., Grabarek Z., Gergely J., Collins J. H. Cross-linking of residue 57 in the regulatory domain of a mutant rabbit skeletal muscle troponin C to the inhibitory region of troponin I. J Biol Chem. 1991 Jul 25;266(21):13746–13751. [PubMed] [Google Scholar]
  22. Krudy G. A., Kleerekoper Q., Guo X., Howarth J. W., Solaro R. J., Rosevear P. R. NMR studies delineating spatial relationships within the cardiac troponin I-troponin C complex. J Biol Chem. 1994 Sep 23;269(38):23731–23735. [PubMed] [Google Scholar]
  23. Leavis P. C., Gergely J. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. CRC Crit Rev Biochem. 1984;16(3):235–305. doi: 10.3109/10409238409108717. [DOI] [PubMed] [Google Scholar]
  24. Leavis P. C., Rosenfeld S., Lu R. C. Cleavage of a specific bond in troponin C by thrombin. Biochim Biophys Acta. 1978 Aug 21;535(2):281–286. doi: 10.1016/0005-2795(78)90094-6. [DOI] [PubMed] [Google Scholar]
  25. Leszyk J., Collins J. H., Leavis P. C., Tao T. Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T. Biochemistry. 1988 Sep 6;27(18):6983–6987. doi: 10.1021/bi00418a047. [DOI] [PubMed] [Google Scholar]
  26. Leszyk J., Collins J. H., Leavis P. C., Tao T. Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C. Biochemistry. 1987 Nov 3;26(22):7042–7047. doi: 10.1021/bi00396a028. [DOI] [PubMed] [Google Scholar]
  27. Leszyk J., Grabarek Z., Gergely J., Collins J. H. Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C. Biochemistry. 1990 Jan 9;29(1):299–304. doi: 10.1021/bi00453a041. [DOI] [PubMed] [Google Scholar]
  28. Li M. X., Spyracopoulos L., Sykes B. D. Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C. Biochemistry. 1999 Jun 29;38(26):8289–8298. doi: 10.1021/bi9901679. [DOI] [PubMed] [Google Scholar]
  29. Luo Y., Leszyk J., Qian Y., Gergely J., Tao T. Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I. Biochemistry. 1999 May 18;38(20):6678–6688. doi: 10.1021/bi9824341. [DOI] [PubMed] [Google Scholar]
  30. Luo Y., Wu J. L., Gergely J., Tao T. Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer. Biophys J. 1998 Jun;74(6):3111–3119. doi: 10.1016/S0006-3495(98)78017-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luo Y., Wu J. L., Gergely J., Tao T. Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments. Biochemistry. 1997 Sep 9;36(36):11027–11035. doi: 10.1021/bi962461w. [DOI] [PubMed] [Google Scholar]
  32. Luo Y., Wu J. L., Li B., Langsetmo K., Gergely J., Tao T. Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins. J Mol Biol. 2000 Feb 25;296(3):899–910. doi: 10.1006/jmbi.1999.3495. [DOI] [PubMed] [Google Scholar]
  33. Malnic B., Farah C. S., Reinach F. C. Regulatory properties of the NH2- and COOH-terminal domains of troponin T. ATPase activation and binding to troponin I and troponin C. J Biol Chem. 1998 Apr 24;273(17):10594–10601. doi: 10.1074/jbc.273.17.10594. [DOI] [PubMed] [Google Scholar]
  34. McKay R. T., Pearlstone J. R., Corson D. C., Gagné S. M., Smillie L. B., Sykes B. D. Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I. Biochemistry. 1998 Sep 8;37(36):12419–12430. doi: 10.1021/bi9809019. [DOI] [PubMed] [Google Scholar]
  35. McKay R. T., Tripet B. P., Hodges R. S., Sykes B. D. Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy. J Biol Chem. 1997 Nov 7;272(45):28494–28500. doi: 10.1074/jbc.272.45.28494. [DOI] [PubMed] [Google Scholar]
  36. McKay R. T., Tripet B. P., Pearlstone J. R., Smillie L. B., Sykes B. D. Defining the region of troponin-I that binds to troponin-C. Biochemistry. 1999 Apr 27;38(17):5478–5489. doi: 10.1021/bi9829736. [DOI] [PubMed] [Google Scholar]
  37. Olah G. A., Rokop S. E., Wang C. L., Blechner S. L., Trewhella J. Troponin I encompasses an extended troponin C in the Ca(2+)-bound complex: a small-angle X-ray and neutron scattering study. Biochemistry. 1994 Jul 12;33(27):8233–8239. doi: 10.1021/bi00193a009. [DOI] [PubMed] [Google Scholar]
  38. Oliveira D. M., Nakaie C. R., Sousa A. D., Farah C. S., Reinach F. C. Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity. Identification of residues involved in binding to actin. J Biol Chem. 2000 Sep 8;275(36):27513–27519. doi: 10.1074/jbc.M002735200. [DOI] [PubMed] [Google Scholar]
  39. Pearlstone J. R., Smillie L. B. Evidence for two-site binding of troponin I inhibitory peptides to the N and C domains of troponin C. Biochemistry. 1995 May 30;34(21):6932–6940. doi: 10.1021/bi00021a004. [DOI] [PubMed] [Google Scholar]
  40. Pearlstone J. R., Smillie L. B. The interaction of rabbit skeletal muscle troponin-T fragments with troponin-I. Can J Biochem Cell Biol. 1985 Mar;63(3):212–218. doi: 10.1139/o85-030. [DOI] [PubMed] [Google Scholar]
  41. Pearlstone J. R., Sykes B. D., Smillie L. B. Interactions of structural C and regulatory N domains of troponin C with repeated sequence motifs in troponin I. Biochemistry. 1997 Jun 17;36(24):7601–7606. doi: 10.1021/bi970200w. [DOI] [PubMed] [Google Scholar]
  42. Perry S. V. Troponin I: inhibitor or facilitator. Mol Cell Biochem. 1999 Jan;190(1-2):9–32. [PubMed] [Google Scholar]
  43. Perry S. V. Troponin T: genetics, properties and function. J Muscle Res Cell Motil. 1998 Aug;19(6):575–602. doi: 10.1023/a:1005397501968. [DOI] [PubMed] [Google Scholar]
  44. Potter J. D., Sheng Z., Pan B. S., Zhao J. A direct regulatory role for troponin T and a dual role for troponin C in the Ca2+ regulation of muscle contraction. J Biol Chem. 1995 Feb 10;270(6):2557–2562. doi: 10.1074/jbc.270.6.2557. [DOI] [PubMed] [Google Scholar]
  45. Sheng Z., Pan B. S., Miller T. E., Potter J. D. Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. J Biol Chem. 1992 Dec 15;267(35):25407–25413. [PubMed] [Google Scholar]
  46. Slupsky C. M., Sykes B. D. NMR solution structure of calcium-saturated skeletal muscle troponin C. Biochemistry. 1995 Dec 12;34(49):15953–15964. doi: 10.1021/bi00049a010. [DOI] [PubMed] [Google Scholar]
  47. Soman J., Tao T., Phillips G. N., Jr Conformational variation of calcium-bound troponin C. Proteins. 1999 Dec 1;37(4):510–511. doi: 10.1002/(sici)1097-0134(19991201)37:4<510::aid-prot2>3.0.co;2-t. [DOI] [PubMed] [Google Scholar]
  48. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  49. Stefancsik R., Jha P. K., Sarkar S. Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: potential role for coiled coil interaction. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):957–962. doi: 10.1073/pnas.95.3.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stone D. B., Timmins P. A., Schneider D. K., Krylova I., Ramos C. H., Reinach F. C., Mendelson R. A. The effect of regulatory Ca2+ on the in situ structures of troponin C and troponin I: a neutron scattering study. J Mol Biol. 1998 Aug 28;281(4):689–704. doi: 10.1006/jmbi.1998.1965. [DOI] [PubMed] [Google Scholar]
  51. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  52. Strynadka N. C., Cherney M., Sielecki A. R., Li M. X., Smillie L. B., James M. N. Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution. J Mol Biol. 1997 Oct 17;273(1):238–255. doi: 10.1006/jmbi.1997.1257. [DOI] [PubMed] [Google Scholar]
  53. Sundaralingam M., Bergstrom R., Strasburg G., Rao S. T., Roychowdhury P., Greaser M., Wang B. C. Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution. Science. 1985 Feb 22;227(4689):945–948. doi: 10.1126/science.3969570. [DOI] [PubMed] [Google Scholar]
  54. Syska H., Wilkinson J. M., Grand R. J., Perry S. V. The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit. Biochem J. 1976 Feb 1;153(2):375–387. doi: 10.1042/bj1530375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Talbot J. A., Hodges R. S. Synthetic studies on the inhibitory region of rabbit skeletal troponin I. Relationship of amino acid sequence to biological activity. J Biol Chem. 1981 Mar 25;256(6):2798–2802. [PubMed] [Google Scholar]
  56. Tao T., Cho J. Fluorescence lifetime quenching studies on the accessibilities of actin sulfhydryl sites. Biochemistry. 1979 Jun 26;18(13):2759–2765. doi: 10.1021/bi00580a011. [DOI] [PubMed] [Google Scholar]
  57. Tao T., Gong B. J., Grabarek Z., Gergely J. Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin. Biochim Biophys Acta. 1999 Jul 8;1450(3):423–433. doi: 10.1016/s0167-4889(99)00050-6. [DOI] [PubMed] [Google Scholar]
  58. Tao T., Gowell E., Strasburg G. M., Gergely J., Leavis P. C. Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements. Biochemistry. 1989 Jul 11;28(14):5902–5908. doi: 10.1021/bi00440a029. [DOI] [PubMed] [Google Scholar]
  59. Tao T., Lamkin M., Lehrer S. S. Excitation energy transfer studies of the proximity between tropomyosin and actin in reconstituted skeletal muscle thin filaments. Biochemistry. 1983 Jun 21;22(13):3059–3066. doi: 10.1021/bi00282a006. [DOI] [PubMed] [Google Scholar]
  60. Tao T., Lamkin M., Scheiner C. J. The conformation of the C-terminal region of actin: a site-specific photocrosslinking study using benzophenone-4-maleimide. Arch Biochem Biophys. 1985 Aug 1;240(2):627–634. doi: 10.1016/0003-9861(85)90070-0. [DOI] [PubMed] [Google Scholar]
  61. Tao T. Nanosecond fluorescence depolarization studies on actin labeled with 1,5-IAEDANS and dansyl chloride. Evidence for label flexibility. FEBS Lett. 1978 Sep 1;93(1):146–150. doi: 10.1016/0014-5793(78)80824-2. [DOI] [PubMed] [Google Scholar]
  62. Tobacman L. S. Thin filament-mediated regulation of cardiac contraction. Annu Rev Physiol. 1996;58:447–481. doi: 10.1146/annurev.ph.58.030196.002311. [DOI] [PubMed] [Google Scholar]
  63. Tripet B., Van Eyk J. E., Hodges R. S. Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction. J Mol Biol. 1997 Sep 5;271(5):728–750. doi: 10.1006/jmbi.1997.1200. [DOI] [PubMed] [Google Scholar]
  64. Van Eyk J. E., Thomas L. T., Tripet B., Wiesner R. J., Pearlstone J. R., Farah C. S., Reinach F. C., Hodges R. S. Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. J Biol Chem. 1997 Apr 18;272(16):10529–10537. doi: 10.1074/jbc.272.16.10529. [DOI] [PubMed] [Google Scholar]
  65. Vassylyev D. G., Takeda S., Wakatsuki S., Maeda K., Maéda Y. Crystal structure of troponin C in complex with troponin I fragment at 2.3-A resolution. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4847–4852. doi: 10.1073/pnas.95.9.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wang Z. Y., Sarkar S., Gergely J., Tao T. Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C. J Biol Chem. 1990 Mar 25;265(9):4953–4957. [PubMed] [Google Scholar]
  67. Wang Z., Gergely J., Tao T. Characterization of the Ca(2+)-triggered conformational transition in troponin C. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11814–11817. doi: 10.1073/pnas.89.24.11814. [DOI] [PMC free article] [PubMed] [Google Scholar]

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