Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Aug;5(8):1531–1540. doi: 10.1002/pro.5560050809

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.

P O Freskgård 1, O H Olsen 1, E Persson 1
PMCID: PMC2143475  PMID: 8844844

Abstract

Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Ashton A. W., Kemball-Cook G., Johnson D. J., Martin D. M., O'Brien D. P., Tuddenham E. G., Perkins S. J. Factor VIIa and the extracellular domains of human tissue factor form a compact complex: a study by X-ray and neutron solution scattering. FEBS Lett. 1995 Oct 23;374(1):141–146. doi: 10.1016/0014-5793(95)01093-t. [DOI] [PubMed] [Google Scholar]
  2. Atkinson R. A., Evans J. S., Hauschka P. V., Levine B. A., Meats R., Triffitt J. T., Virdi A. S., Williams R. J. Conformational studies of osteocalcin in solution. Eur J Biochem. 1995 Sep 1;232(2):515–521. doi: 10.1111/j.1432-1033.1995.tb20838.x. [DOI] [PubMed] [Google Scholar]
  3. Banner D. W., D'Arcy A., Chène C., Winkler F. K., Guha A., Konigsberg W. H., Nemerson Y., Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996 Mar 7;380(6569):41–46. doi: 10.1038/380041a0. [DOI] [PubMed] [Google Scholar]
  4. Brandstetter H., Bauer M., Huber R., Lollar P., Bode W. X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9796–9800. doi: 10.1073/pnas.92.21.9796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brandstetter H., Turk D., Hoeffken H. W., Grosse D., Stürzebecher J., Martin P. D., Edwards B. F., Bode W. Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics. J Mol Biol. 1992 Aug 20;226(4):1085–1099. doi: 10.1016/0022-2836(92)91054-s. [DOI] [PubMed] [Google Scholar]
  6. Cascio M., Wallace B. A. Effects of local environment on the circular dichroism spectra of polypeptides. Anal Biochem. 1995 May 1;227(1):90–100. doi: 10.1006/abio.1995.1257. [DOI] [PubMed] [Google Scholar]
  7. Chang J. Y., Stafford D. W., Straight D. L. The roles of factor VII's structural domains in tissue factor binding. Biochemistry. 1995 Sep 26;34(38):12227–12232. doi: 10.1021/bi00038a017. [DOI] [PubMed] [Google Scholar]
  8. Christiansen W. T., Jalbert L. R., Robertson R. M., Jhingan A., Prorok M., Castellino F. J. Hydrophobic amino acid residues of human anticoagulation protein C that contribute to its functional binding to phospholipid Vesicles. Biochemistry. 1995 Aug 22;34(33):10376–10382. doi: 10.1021/bi00033a008. [DOI] [PubMed] [Google Scholar]
  9. Clarke B. J., Ofosu F. A., Sridhara S., Bona R. D., Rickles F. R., Blajchman M. A. The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor. FEBS Lett. 1992 Feb 24;298(2-3):206–210. doi: 10.1016/0014-5793(92)80058-o. [DOI] [PubMed] [Google Scholar]
  10. Freskgård P. O., Mårtensson L. G., Jonasson P., Jonsson B. H., Carlsson U. Assignment of the contribution of the tryptophan residues to the circular dichroism spectrum of human carbonic anhydrase II. Biochemistry. 1994 Nov 29;33(47):14281–14288. doi: 10.1021/bi00251a041. [DOI] [PubMed] [Google Scholar]
  11. Furie B., Furie B. C. Spectral changes in bovine factor X associated with activation by the venom coagulant protein of Vipera russelli. J Biol Chem. 1976 Nov 10;251(21):6807–6814. [PubMed] [Google Scholar]
  12. Gagné S. M., Tsuda S., Li M. X., Chandra M., Smillie L. B., Sykes B. D. Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C. Protein Sci. 1994 Nov;3(11):1961–1974. doi: 10.1002/pro.5560031108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagen F. S., Gray C. L., O'Hara P., Grant F. J., Saari G. C., Woodbury R. G., Hart C. E., Insley M., Kisiel W., Kurachi K. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2412–2416. doi: 10.1073/pnas.83.8.2412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harlos K., Martin D. M., O'Brien D. P., Jones E. Y., Stuart D. I., Polikarpov I., Miller A., Tuddenham E. G., Boys C. W. Crystal structure of the extracellular region of human tissue factor. Nature. 1994 Aug 25;370(6491):662–666. doi: 10.1038/370662a0. [DOI] [PubMed] [Google Scholar]
  15. Hennessey J. P., Jr, Johnson W. C., Jr Experimental errors and their effect on analyzing circular dichroism spectra of proteins. Anal Biochem. 1982 Sep 1;125(1):177–188. doi: 10.1016/0003-2697(82)90400-6. [DOI] [PubMed] [Google Scholar]
  16. Higashi S., Nishimura H., Aita K., Iwanaga S. Identification of regions of bovine factor VII essential for binding to tissue factor. J Biol Chem. 1994 Jul 22;269(29):18891–18898. [PubMed] [Google Scholar]
  17. Higashi S., Nishimura H., Fujii S., Takada K., Iwanaga S. Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate. J Biol Chem. 1992 Sep 5;267(25):17990–17996. [PubMed] [Google Scholar]
  18. Hirst J. D., Brooks C. L., 3rd Helicity, circular dichroism and molecular dynamics of proteins. J Mol Biol. 1994 Oct 21;243(2):173–178. doi: 10.1006/jmbi.1994.1644. [DOI] [PubMed] [Google Scholar]
  19. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  20. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  21. Kazama Y., Pastuszyn A., Wildgoose P., Hamamoto T., Kisiel W. Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa. J Biol Chem. 1993 Aug 5;268(22):16231–16240. [PubMed] [Google Scholar]
  22. Kelley R. F., Costas K. E., O'Connell M. P., Lazarus R. A. Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding. Biochemistry. 1995 Aug 22;34(33):10383–10392. doi: 10.1021/bi00033a009. [DOI] [PubMed] [Google Scholar]
  23. Keyt B., Furie B. C., Furie B. Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies. J Biol Chem. 1982 Aug 10;257(15):8687–8695. [PubMed] [Google Scholar]
  24. Kumar A., Fair D. S. Specific molecular interaction sites on factor VII involved in factor X activation. Eur J Biochem. 1993 Oct 15;217(2):509–518. doi: 10.1111/j.1432-1033.1993.tb18271.x. [DOI] [PubMed] [Google Scholar]
  25. Li M. X., Chandra M., Pearlstone J. R., Racher K. I., Trigo-Gonzalez G., Borgford T., Kay C. M., Smillie L. B. Properties of isolated recombinant N and C domains of chicken troponin C. Biochemistry. 1994 Feb 1;33(4):917–925. doi: 10.1021/bi00170a010. [DOI] [PubMed] [Google Scholar]
  26. Manavalan P., Johnson W. C., Jr Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem. 1987 Nov 15;167(1):76–85. doi: 10.1016/0003-2697(87)90135-7. [DOI] [PubMed] [Google Scholar]
  27. Manning M. C., Illangasekare M., Woody R. W. Circular dichroism studies of distorted alpha-helices, twisted beta-sheets, and beta turns. Biophys Chem. 1988 Aug;31(1-2):77–86. doi: 10.1016/0301-4622(88)80011-5. [DOI] [PubMed] [Google Scholar]
  28. Manning M. C., Woody R. W. Theoretical study of the contribution of aromatic side chains to the circular dichroism of basic bovine pancreatic trypsin inhibitor. Biochemistry. 1989 Oct 17;28(21):8609–8613. doi: 10.1021/bi00447a051. [DOI] [PubMed] [Google Scholar]
  29. Marsh H. C., Robertson P., Jr, Scott M. E., Koehler K. A., Hiskey R. G. Magnesium and calcium ion binding to bovine prothrombin fragment 1. A circular dichroism, fluorescence, and 43Ca2+ and 25Mg2+ nuclear magnetic resonance study. J Biol Chem. 1979 Oct 25;254(20):10268–10275. [PubMed] [Google Scholar]
  30. Martin D. M., Boys C. W., Ruf W. Tissue factor: molecular recognition and cofactor function. FASEB J. 1995 Jul;9(10):852–859. doi: 10.1096/fasebj.9.10.7615155. [DOI] [PubMed] [Google Scholar]
  31. Medved L. V., Orthner C. L., Lubon H., Lee T. K., Drohan W. N., Ingham K. C. Thermal stability and domain-domain interactions in natural and recombinant protein C. J Biol Chem. 1995 Jun 9;270(23):13652–13659. doi: 10.1074/jbc.270.23.13652. [DOI] [PubMed] [Google Scholar]
  32. Medved L. V., Vysotchin A., Ingham K. C. Ca(2+)-dependent interactions between Gla and EGF domains in human coagulation factor IX. Biochemistry. 1994 Jan 18;33(2):478–485. doi: 10.1021/bi00168a012. [DOI] [PubMed] [Google Scholar]
  33. Muller Y. A., Ultsch M. H., Kelley R. F., de Vos A. M. Structure of the extracellular domain of human tissue factor: location of the factor VIIa binding site. Biochemistry. 1994 Sep 13;33(36):10864–10870. doi: 10.1021/bi00202a003. [DOI] [PubMed] [Google Scholar]
  34. Nelsestuen G. L., Resnick R. M., Wei G. J., Pletcher C. H., Bloomfield V. A. Metal ion interactions with bovine prothrombin and prothrombin fragment 1. Stoichiometry of binding, protein self-association, and conformational change induced by a variety of metal ions. Biochemistry. 1981 Jan 20;20(2):351–358. doi: 10.1021/bi00505a019. [DOI] [PubMed] [Google Scholar]
  35. Owen W. G., Bichler J., Ericson D., Wysokinski W. Gating of thrombin in platelet aggregates by pO2-linked lowering of extracellular Ca2+ concentration. Biochemistry. 1995 Jul 25;34(29):9277–9281. doi: 10.1021/bi00029a001. [DOI] [PubMed] [Google Scholar]
  36. Padmanabhan K., Padmanabhan K. P., Tulinsky A., Park C. H., Bode W., Huber R., Blankenship D. T., Cardin A. D., Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol. 1993 Aug 5;232(3):947–966. doi: 10.1006/jmbi.1993.1441. [DOI] [PubMed] [Google Scholar]
  37. Persson E. Influence of the gamma-carboxyglutamic acid-rich domain and hydrophobic stack of factor VIIa on tissue factor binding. Haemostasis. 1996;26 (Suppl 1):31–34. doi: 10.1159/000217237. [DOI] [PubMed] [Google Scholar]
  38. Persson E., Petersen L. C. Structurally and functionally distinct Ca2+ binding sites in the gamma-carboxyglutamic acid-containing domain of factor VIIa. Eur J Biochem. 1995 Nov 15;234(1):293–300. doi: 10.1111/j.1432-1033.1995.293_c.x. [DOI] [PubMed] [Google Scholar]
  39. Persson E., Valcarce C., Stenflo J. The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X. J Biol Chem. 1991 Feb 5;266(4):2453–2458. [PubMed] [Google Scholar]
  40. Petersen L. C., Schiødt J., Christensen U. Involvement of the hydrophobic stack residues 39-44 of factor VIIa in tissue factor interactions. FEBS Lett. 1994 Jun 20;347(1):73–79. doi: 10.1016/0014-5793(94)00513-3. [DOI] [PubMed] [Google Scholar]
  41. Ruf W., Edgington T. S. Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8430–8434. doi: 10.1073/pnas.88.19.8430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ruf W., Kalnik M. W., Lund-Hansen T., Edgington T. S. Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions. J Biol Chem. 1991 Aug 25;266(24):15719–15725. [PubMed] [Google Scholar]
  43. Sabharwal A. K., Birktoft J. J., Gorka J., Wildgoose P., Petersen L. C., Bajaj S. P. High affinity Ca(2+)-binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity. J Biol Chem. 1995 Jun 30;270(26):15523–15530. doi: 10.1074/jbc.270.26.15523. [DOI] [PubMed] [Google Scholar]
  44. Sakai T., Lund-Hansen T., Thim L., Kisiel W. The gamma-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor. J Biol Chem. 1990 Feb 5;265(4):1890–1894. [PubMed] [Google Scholar]
  45. Schiødt J., Harrit N., Christensen U., Petersen L. C. Two different Ca2+ ion binding sites in factor VIIa and in des(1-38) factor VIIa. FEBS Lett. 1992 Jul 20;306(2-3):265–268. doi: 10.1016/0014-5793(92)81014-d. [DOI] [PubMed] [Google Scholar]
  46. Selander-Sunnerhagen M., Ullner M., Persson E., Teleman O., Stenflo J., Drakenberg T. How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X. J Biol Chem. 1992 Sep 25;267(27):19642–19649. doi: 10.2210/pdb1ccf/pdb. [DOI] [PubMed] [Google Scholar]
  47. Soriano-Garcia M., Padmanabhan K., de Vos A. M., Tulinsky A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry. 1992 Mar 10;31(9):2554–2566. doi: 10.1021/bi00124a016. [DOI] [PubMed] [Google Scholar]
  48. Stone M. J., Ruf W., Miles D. J., Edgington T. S., Wright P. E. Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization. Biochem J. 1995 Sep 1;310(Pt 2):605–614. doi: 10.1042/bj3100605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sugo T., Björk I., Holmgren A., Stenflo J. Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region. J Biol Chem. 1984 May 10;259(9):5705–5710. [PubMed] [Google Scholar]
  50. Sunnerhagen M., Forsén S., Hoffrén A. M., Drakenberg T., Teleman O., Stenflo J. Structure of the Ca(2+)-free Gla domain sheds light on membrane binding of blood coagulation proteins. Nat Struct Biol. 1995 Jun;2(6):504–509. doi: 10.1038/nsb0695-504. [DOI] [PubMed] [Google Scholar]
  51. Thim L., Bjoern S., Christensen M., Nicolaisen E. M., Lund-Hansen T., Pedersen A. H., Hedner U. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry. 1988 Oct 4;27(20):7785–7793. doi: 10.1021/bi00420a030. [DOI] [PubMed] [Google Scholar]
  52. Toomey J. R., Smith K. J., Stafford D. W. Localization of the human tissue factor recognition determinant of human factor VIIa. J Biol Chem. 1991 Oct 15;266(29):19198–19202. [PubMed] [Google Scholar]
  53. Toumadje A., Alcorn S. W., Johnson W. C., Jr Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures. Anal Biochem. 1992 Feb 1;200(2):321–331. doi: 10.1016/0003-2697(92)90473-k. [DOI] [PubMed] [Google Scholar]
  54. Valcarce C., Holmgren A., Stenflo J. Calcium-dependent interaction between gamma-carboxyglutamic acid-containing and N-terminal epidermal growth factor-like modules in factor X. J Biol Chem. 1994 Oct 21;269(42):26011–26016. [PubMed] [Google Scholar]
  55. Vuilleumier S., Sancho J., Loewenthal R., Fersht A. R. Circular dichroism studies of barnase and its mutants: characterization of the contribution of aromatic side chains. Biochemistry. 1993 Oct 5;32(39):10303–10313. doi: 10.1021/bi00090a005. [DOI] [PubMed] [Google Scholar]
  56. Vysotchin A., Medved L. V., Ingham K. C. Domain structure and domain-domain interactions in human coagulation factor IX. J Biol Chem. 1993 Apr 25;268(12):8436–8446. [PubMed] [Google Scholar]
  57. Wildgoose P., Foster D., Schiødt J., Wiberg F. C., Birktoft J. J., Petersen L. C. Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII-tissue factor interaction. Biochemistry. 1993 Jan 12;32(1):114–119. doi: 10.1021/bi00052a016. [DOI] [PubMed] [Google Scholar]
  58. Wildgoose P., Jørgensen T., Komiyama Y., Nakagaki T., Pedersen A., Kisiel W. The role of phospholipids and the factor VII Gla-domain in the interaction of factor VII with tissue factor. Thromb Haemost. 1992 Jun 1;67(6):679–685. [PubMed] [Google Scholar]
  59. Wildgoose P., Kazim A. L., Kisiel W. The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7290–7294. doi: 10.1073/pnas.87.18.7290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Woody R. W. Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins. Eur Biophys J. 1994;23(4):253–262. doi: 10.1007/BF00213575. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES