Use of Protein Chemistry and Molecular Biology to Determine Interaction Areas Between Proteases and their Inhibitors: The Thrombin-Hirudin Interaction as an Example

  • Stuart R. Stone
  • Stanley Dennis
  • Andrew Wallace
  • Jan Hofsteenge
Part of the NATO ASI Series book series (NSSA, volume 191)

Abstract

Thrombin is a serine protease that exhibits the same primary specificity as trypsin, i.e., it cleaves peptide bonds on the C-terminal side of basic amino acids (preferably arginine) Thrombin, however, exhibits a narrower specificity than trypsin with respect to the peptide bonds that it will cleave. This narrower specificity of thrombin is partly due to the presence on thrombin of secondary binding sites and this article will concentrate on the contribution of these sites to the formation of the complex between thrombin and the inhibitor hirudin. After a brief introduction on thrombin, hirudin and the kinetics of the formation of their complex, studies aimed at identifying secondary binding sites for hirudin on thrombin will be considered. These studies have used mainly the techniques of protein chemistry. The last part of the article will consider the use of site-directed mutagenesis to elucidate areas of hirudin important for its inhibitory activity.

Keywords

Serine Lysine Arginine Glutamine Trypsin 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    K.G. Mann, The assembly of blood clotting complexes on membranes. Trends Biochem. Sci. 12: 229 (1987).Google Scholar
  2. 2.
    J.W. Fenton, Structural regions and bioregulatory functions of thrombin, in: “Cell proliferation: Recent advances”, Vol. II, Boynton, A. L., and Leffert, H. L., eds., Academic Press, New York (1987).Google Scholar
  3. 3.
    R. Bar-Shavit, A. Kahn, J.W. Fenton, II, and G.D. Wilner, Chemotactic response of monocytes to thrombin. J. Cell Biol. 96: 282 (1983).CrossRefGoogle Scholar
  4. 4.
    R. Bar-Shavit, A. Kahn, G.D. Wilner, and J.W. Fenton, II, Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 220: 728 (1983).PubMedCrossRefGoogle Scholar
  5. 5.
    L.B. Chen, and J.M. Buchanan, Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc. Natl. Acad. Sci. U.S.A. 72: 131 (1975).CrossRefGoogle Scholar
  6. 6.
    J.W. Fenton, II, Thrombin specificity. Ann. N. Y. Acad. Sci. 370: 468 (1981).PubMedCrossRefGoogle Scholar
  7. 7.
    J.W. Fenton, II, and D.H. Bing, Thrombin active-site regions. Semin. Thromb. Hemostasis 12: 200 (1986).CrossRefGoogle Scholar
  8. 8.
    J.B. Haycraft, Secretion obtained from the medicinal leech. Proc. R. Soc. London 36B: 478 (1884).Google Scholar
  9. F. Markwardt, Die Isolierung und chemische Charakterisierung des Hirudins. Hoppe-Seylers Z. Physiol. Chem. 308: 147 (1957).Google Scholar
  10. 10.
    D. Bagdy, E. Barabas, L. Graf, T.E. Peterson, and S. Magnusson, Hirudin. Methods Enzymol. 45: 669 (1976).CrossRefGoogle Scholar
  11. 11.
    J. Dodt, H. Müller, U. Seemüller, and J.-Y. Chang, The complete amino acid sequence of hirudin, a thrombin specific inhibitor. FEBS Lett. 165: 180 (1984).CrossRefGoogle Scholar
  12. 12.
    S.J.T. Mao, M.T. Yates, D.T. Blankenship, A.D. Cardin, J.L. Krstenansky, W. Lovenberg, and R.L. Jackson, Rapid purification and revised N-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech. Anal. Biochem. 161: 514 (1987).PubMedCrossRefGoogle Scholar
  13. 13.
    J. Dodt, U. Seemüller, R. Maschler, and H. Fritz, The complete covalent structure of hirudin. Localisation of the disulfide bonds. Biol. Chem. Hoppe-Seyler 366: 379 (1985).PubMedCrossRefGoogle Scholar
  14. 14.
    J. Dodt, N. Machleidt, U. Seemüller, R. Maschler, and H. Fritz, Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA. Biol. Chem. Hoppe-Seyler 367: 803 (1986).PubMedCrossRefGoogle Scholar
  15. 15.
    J. Dodt, T. Schmitz, T. Schäfer, and C. Bergmann, Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. FEBS Lett. 202: 373 (1986).PubMedCrossRefGoogle Scholar
  16. 16.
    R.P. Harvey, E. Degryse, L. Stefani, F. Schamber, J.-P. Cazenave, M. Courtney, P. Tolstoshev, and J.-P. Lecocq, Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis. Proc. Natl. Acad. Sci. USA 83: 1084 (1986).PubMedCrossRefGoogle Scholar
  17. 17.
    G.M. Clore, D.K. Sukumaran, M. Nilges, J. Zarbock, and A.M. Gronenborn, The conformations of hirudin in solution: a study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics. EMBO J. 6: 529 (1987).Google Scholar
  18. 18.
    D.K. Sukumaran, G.M. Clore, A. Preuss, J. Zarbock, and A.M. Gronenborn, Proton nuclear magnetic resonance study of hirudin: resonance assignment and secondary structure. Biochemistry 26: 333 (1987).CrossRefGoogle Scholar
  19. 19.
    P.J.M. Folkers, G.M. Clore, P.C. Driscoll, J. Dodt, S. Köhler, and A.M. Gronenborn, Solution structure of recombinant hirudin and the Lys-47 - Glu mutant: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry 28: 2601 (1989).PubMedCrossRefGoogle Scholar
  20. 20.
    H. Haruyama, and K. Wüthrich, The conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry 28: 4301 (1989).PubMedCrossRefGoogle Scholar
  21. 21.
    H. Haruyama, Y.-Q. Qian, and K. Wüthrich, Static and transient hydrogen bonding interactions in recombinant desulfatohirudin studied by 1H nuclear magnetic resonance measurements of amide proton exchange rates and pH dependent chemical shifts. Biochemistry 28: 4312 (1989).PubMedCrossRefGoogle Scholar
  22. 22.
    S.R. Stone, and J. Hofsteenge, Kinetics of the inhibition of thrombin by hirudin. Biochemistry 25: 4622 (1986).PubMedCrossRefGoogle Scholar
  23. 23.
    P.J. Braun, S. Dennis, J. Hofsteenge, and S.R. Stone, Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin Biochemistry 27: 6517 (1988).Google Scholar
  24. 24.
    R.B. Wallis, Hirudins and the role of thrombin: lessons from leeches. Trends Pharmac. Sci. 9: 425 (1988).Google Scholar
  25. 25.
    F. Markwardt, and P. Walsmann, Die Reaktion zwischen Hirudin und Thrombin. Hoppe-Seylers Z. Physiol. Chem. 312: 85 (1958).CrossRefGoogle Scholar
  26. 26.
    B.H. Landis, M.P. Zabinski, G.J.M. Lafleur, D.H. Bing, and J.W. Fenton, II, Human a-thrombin and 7-thrombin differential inhibition with hirudin. Fed. Proc. 37: 1445 (1978).Google Scholar
  27. 27.
    S.R. Stone, S. Dennis, and J. Hofsteenge, Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex. Biochemistry in press (1989).Google Scholar
  28. 28.
    W.G. Landis, and D.F. Waugh, Interactions of bovine fibrinogen and thrombin. Early events in the development of clot structure. Arch. Biochem. Biophys. 168: 498 (1975).PubMedCrossRefGoogle Scholar
  29. 29.
    J. Dodt, S. Köhler, and A. Baici, Interaction of site specific hirudin variants with a-thrombin. FEBS Lett. 229: 87 (1988).PubMedCrossRefGoogle Scholar
  30. 30.
    J.F. Morrison, and C.T. Walsh, The behavior and significance of slow-binding enzyme inhibitors. Adv. Enzymol. and Rel. Areas Mol. Biol. 61: 201 (1987).Google Scholar
  31. 31.
    S.R. Stone, P.J. Braun, and J. Hofsteenge, Identification of regions of a-thrombin involved in its interaction with hirudin. Biochemistry 26: 4617 (1987).PubMedCrossRefGoogle Scholar
  32. 32.
    L.J. Berliner, and Y.Y.L. Shen, Physical evidence for an apolar binding site near the catalytic center of human a-thrombin. Biochemistry 16: 4622 (1977).PubMedCrossRefGoogle Scholar
  33. 33.
    G. Claeson, L. Aurell, G. Karlsson, and P. Friberger, Substrate structure and activity relationship, in: “New methods for analysis of coagulation using chromogenic substrates”, Witt, I., ed., de Gruyter, Berlin (1977).Google Scholar
  34. 34.
    C. Kettner, and E. Shaw, Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone. Methods Enzymol. 80: 826 (1981).PubMedCrossRefGoogle Scholar
  35. 35.
    G. Glover, and E. Shaw, The purification of thrombin and isolation of a peptide containing the active center histidine. J. Biol. Chem. 246: 4594 (1971).Google Scholar
  36. 36.
    SA. Sonder, and J.W. Fenton, II, Proflavin binding within the fibrinopeptide groove adjacent to the catalytic site of human a-thrombin. Biochemistry 23: 1818 (1984).PubMedCrossRefGoogle Scholar
  37. 37.
    B. Walker, P. Wikström, and E. Shaw, Evaluation of inhibitor constants and alkylation rates for a series of thrombin affinity labels. Biochem. J. 230: 645 (1985).PubMedGoogle Scholar
  38. 38.
    J.W. Fenton, II, B.H. Landis, D.A. Walz, D.H. Bing, R.D. Feinman, M.P. Zabinski, SA. Sonder, L.J. Berliner, and J.S. Finlayson, Human thrombin: preparative evaluation, structural properties, and enzymic specificity, in: “The chemistry and physiology of human plasma proteins”, Bing, D. H., ed., Pergamon Press, New York (1979).Google Scholar
  39. 39.
    L.J. Berliner, Structure-functions relationships in human a-and 1-thrombin. Mol. Cell. Biochem. 61: 159 (1984).PubMedCrossRefGoogle Scholar
  40. 40.
    J.W. Fenton, II, B.H. Landis, DA Walz, and J.S. Finlayson, Human thrombins, in: “Chemistry and biology of thrombin”, Lundblad; R. L., Fenton, J. W., II, and Mann, K. G., eds., Ann Arbor Science, Ann Arbor (1977).Google Scholar
  41. 41.
    J.-P. Boissel, B. Le Bonniec, M.-J. Rabiet, D. Labie, and J. Elion, Covalent structure of ß-and 7 autolytic derivatives of human a-thrombin. J. Biol. Chem. 259: 5691 (1984).Google Scholar
  42. 42.
    J.-Y. Chang, The structures and proteolytic specificities of autolyzed human thrombin. Biochem. J. 240: 797 (1986).PubMedGoogle Scholar
  43. 43.
    P.J. Braun, J. Hofsteenge, J.-Y. Chang, and S.R. Stone, Preparation and characterization of proteolyzed forms of human a-thrombin. Thromb. Res. 50: 273 (1988).Google Scholar
  44. R. Lottenberg, JA. Hall, J.W. Fenton, II, and C.M. Jackson, The action of thrombin on peptide pnitroanilide substrates; hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human a and 1 and bovine a and ß-thrombins. Thromb. Res. 28: 313 (1982).Google Scholar
  45. 45.
    SA. Sonder, and J.W. Fenton, II, Thrombin specificity with tripeptide chromogenic substrates: comparison of human and bovine thrombins with and without fibrinogen clotting activities. Clin. Chem. 32: 934 (1986).PubMedGoogle Scholar
  46. 46.
    J. Hofsteenge, P.J. Braun, and S.R. Stone, Enzymatic properties of proteolytic derivatives of human a-thrombin. Biochemistry 27: 2144 (1988).PubMedCrossRefGoogle Scholar
  47. 47.
    S.D. Lewis, L. Lorand, J.W. Fenton, II, and JA. Shafer, Catalytic competence of human a-and -y-thrombin in the activation of fibrinogen and factor XIII. Biochemistry 26: 7597 (1987).PubMedCrossRefGoogle Scholar
  48. 48.
    S. Kawabata, T. Morita, S. Iwanaga, and H. Igarashi, Staphylocoagulase-binding region in human prothrombin. J. Biochem. (Tokyo) 97: 325 (1985).Google Scholar
  49. 49.
    G. Noé, J. Hofsteenge, G. Rovelli, and S.R. Stone, The use of sequence specific antibodies to identify a secondary binding site in thrombin. J. Biol. Chem. 263: 11729 (1988).PubMedGoogle Scholar
  50. 50.
    J.-Y. Chang, The hirudin-binding site of human a-thrombin. J. Biol. Chem. 264: 7141 (1989).PubMedGoogle Scholar
  51. 51.
    S. Dennis, A. Wallace, J. Hofsteenge, and S.R. Stone, Use of fragments of hirudin to investigate the thrombin-hirudin interaction. Eur. J. Biochem. in press (1989).Google Scholar
  52. 52.
    J.L. Krstenansky, and S.J.T. Mao, Antithrombin properties of C terminus of hirudin using synthetic unsulfated Na-acetyl-hirudin45–65. FEBS Lett. 211: 10 (1987).PubMedCrossRefGoogle Scholar
  53. 53.
    J.L. Krstenansky, T.J. Owen, M.T. Yates, and S.J.T. Mao, Anticoagulant peptides: Nature of the interaction of the C-terminal region of hirudin with a noncatalytic binding site on thrombin. J. Med. Chem. 30: 1688 (1987).PubMedCrossRefGoogle Scholar
  54. 54.
    S.J.T. Mao, M.T. Yates, T.J. Owen, and J.L. Krstenansky, Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity. Biochemistry 27: 8170 (1988).PubMedCrossRefGoogle Scholar
  55. 55.
    C. Bergmann, J. Dodt, S. Köhler, E. Fink, and H.G. Gassen, Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis. Biol. Chem. Hoppe-Seyler 367: 731 (1986).PubMedCrossRefGoogle Scholar
  56. 56.
    B. Meyhack, J. Heim, H. Rink, W. Zimmermann, and W. Märki, Desulfatohirudin, a specific thrombin inhibitor: expression and secretion in yeast. Thromb. Res. Suppl. 7: 3 (1987).Google Scholar
  57. 57.
    G. Loison, A. Findeli, S. Bernard, M. Nguyen-Juilleret, M. Marquet, N. Riehl-Bellon, D. Cavallo, L. Guerra-Santos, S.W. Brown, M. Courtney, C. Roitsch, and Y. Lemoine, Expression and secretion in S. cerevisiae of biologically active leech hirudin. Biotechnology 6: 72 (1988).CrossRefGoogle Scholar
  58. 58.
    E. Degryse, M. Acker, G. Defreyn, A. Bernat, J.P. Maffrand, C. Roitsch, and M. Courtney, Point mutations modifying the thrombin inhibition kinetics and antithrombin activity in vivo of recombinant hirudin. Protein Eng. 2: 459 (1989).PubMedCrossRefGoogle Scholar
  59. 59.
    A.J. Barret, The cystatins: a new class of peptidase inhibitors. Trends Biochem. Sci. 12: 193 (1986).Google Scholar
  60. 60.
    R.J. Read, and M.N.G. James, Introduction to the proteinase inhibitors: X-ray crystallography, in: “Proteinase Inhibitors” Barret, A. J., and Salvesen, G, eds., Elsevier, Amsterdam (1986).Google Scholar
  61. 61.
    M. Laskowski, Jr., I. Kato, W. Ardelt, J. Cook, A. Denton, M.W. Empie, W.J. Kohr, S.J. Park, K. Parks, B.L. Schatzley, O.L. Schoenberger, M. Tashiro, G. Vichot, H.E. Whatley, A. Wieczorek, and M. Wieczorek, Ovomucoid third domains from 100 avian species: Isolation, sequences, and hypervariability of enzyme-inhibitor contact residues. Biochemistry 26: 202 (1987).Google Scholar
  62. 62.
    M.C. Owen, S.O. Brennan, J H Lewis, and R.W. Carrell, Mutation of antitrypsin to antithrombin. 01-Antitrypsin Pittsburh (358 Met - Arg), a fatal bleeding disorder. N. Engl. J. Med. 309: 694 (1983).PubMedCrossRefGoogle Scholar
  63. 63.
    S. Jallat, D. Carvallo, L.H. Tessier, D. Roecklin, C. Roitsch, F. Ogushi, R.G. Crystal, and M. Courtney, Altered specificities of genetically engineered a1-antitrypsin variants. Protein Eng. 1: 29 (1986).PubMedCrossRefGoogle Scholar
  64. 64.
    J -Y Chang, The functional domain of hirudin, a thrombin-specific inhibitor. FEBS Lett. 164: 307 (1983).PubMedCrossRefGoogle Scholar
  65. 65.
    J. Hofsteenge, S.R. Stone, A. Donella-Deana, and LA. Pinna, The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin. Eur. J. Biochem. in press (1989).Google Scholar
  66. 66.
    A. Wallace, S. Dennis, J. Hofsteenge, and S.R. Stone, Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry in press (1989).Google Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Stuart R. Stone
    • 1
  • Stanley Dennis
    • 1
  • Andrew Wallace
    • 1
  • Jan Hofsteenge
    • 1
  1. 1.Friedrich Miescher-InstitutBaselSwitzerland

Personalised recommendations