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Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1

Nature Structural Biology volume 2, pages 891–897 (1995)Cite this article

Abstract

Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new β-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into β-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation.

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References

  1. Pannekoek, H. et al. Endothelial plasminogen activator inhibitor (PAI): a new member of the serpin gene family. EMBO J. 5, 2539–2544 (1986).

    Article  CAS  Google Scholar 

  2. Ny, T., Sawdey, M., Lawrence, D., Millan, J.L. & Loskutoff, D.J. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc. natn. Acad. Sci. U.S.A. 83, 6776–6780 (1986).

    Article  CAS  Google Scholar 

  3. Ginsburg, D. et al. cDNA cloning of human plasminogen activator inhibitor from endothelial cells. J. clin. Invest. 78, 1673–1680 (1986).

    Article  CAS  Google Scholar 

  4. Andreasen, P.A. et al. Plasminogen activator inhibitor type-1 : reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. FEBS Lett. 209, 213–218 (1986).

    Article  CAS  Google Scholar 

  5. Van Mourik, J.A., Lawrence, D.A. & Loskutoff, D.J. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J. biol. Chem. 259, 14914–14921 (1984).

    CAS  PubMed  Google Scholar 

  6. Sprengers, E.D. & Kluft, C. Plasminogen activator inhibitors. Blood 69, 381–387 (1987).

    CAS  PubMed  Google Scholar 

  7. Alessi, M.C., Declerck, P.J., De Mol, M., Nelles, L. & Collen, D. Purification and characterisation of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1). Eur. J. Biochem. 175, 531–540 (1988).

    Article  CAS  Google Scholar 

  8. Loskutoff, D.J., Sawdey, M. & Mimuro, J. Type 1 plasminogen activator inhibitor. Hemostasis and Thrombosis 9, 87–115 (1989).

    CAS  PubMed  Google Scholar 

  9. Danø, K. et al. Plasminogen activators, tissue degradation, and cancer. Adv. Cancer Res. 44, 139–266 (1985).

    Article  Google Scholar 

  10. Huber, R. & Carrell, R.W. Implications of the three-dimensional structure of α1-antitrypsin for structure and function of serpins. Biochemistry 28, 8951–8966 (1989).

    Article  CAS  Google Scholar 

  11. Laskowski, M. & Kato, I. Protein inhibitors of proteinases. A. Rev. Biochem. 49, 593–626 (1980).

    Article  CAS  Google Scholar 

  12. Stein, P.E. & Chothia, C. Serpin tertiary structure transformation. J. molec. Biol. 221, 615–621 (1991).

    Article  CAS  Google Scholar 

  13. Loebermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. Human α1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J. molec. Biol. 177, 531–556 (1984).

    Article  CAS  Google Scholar 

  14. Wright, H.T., Qian, H.X. & Huber, R. Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved α1-proteinase Inhibitor. J. molec. Biol. 213, 513–528 (1990).

    Article  CAS  Google Scholar 

  15. Stein, P.E., Tewkesbury, D.A. & Carrell, R.W. Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem. J. 262, 661–663 (1989).

    Article  Google Scholar 

  16. Declerck, P.J., De Mol, M., Vaughan, D.E. & Collen, D. Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a non-inhibitory substrate for tissue-type plasminogen activator. J. biol. Chem. 267, 11693–11696 (1992).

    CAS  PubMed  Google Scholar 

  17. Hekman, C.M. & Loskutoff, D.J. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J. biol. Chem. 260, 11581–11587 (1985).

    CAS  PubMed  Google Scholar 

  18. Urano, T., Strandberg, L., Johansson, L.B.-A & Ny, T. A substrate-like form of plasminogen activator inhibitor type 1. Conversion between different forms by sodium dodecyl sulphate. Eur. J. Biochem. 209, 985–992 (1992).

    Article  CAS  Google Scholar 

  19. Munck, M., Heegaard, C.W. & Andreasen, P.A. Interconversions between active, inert and substrate form of denatured/refolded type 1 plasminogen activator inhibitor. Biochim. biophys. Acta 1202, 29–37 (1993).

    Article  Google Scholar 

  20. Vaughan, D.E., Declerck, P.J., Van Houtte, E., De Mol, M. & Collen, D. Studies of recombinant plasminogen activator inhibitor-1 in rabbits : pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo. Circulation Res. 67, 1281–1286 (1990).

    Article  CAS  Google Scholar 

  21. Mottonen, J. et al. Structural basis of latency in plasminogen activator inhibitor-1. Nature 355, 270–273 (1992).

    Article  CAS  Google Scholar 

  22. Carrell, R.W., Evans, D.LI. & Stein, P.E. Mobile reactive centre of serpins Nature 353, 576–578 (1991).

    Article  CAS  Google Scholar 

  23. Audenaert, A.-M., Knockaert, I., Collen, D. & Declerck, P.J. Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop. J. biol. Chem. 269, 19559–19564 (1994).

    CAS  PubMed  Google Scholar 

  24. Perry, D.J., Harper, P.L., Fairham, S., Daly, M. & Carrell, R.W. Antithrombin Cambridge, 384 alanine to Pro: a new variant identified using the polymerase chain reaction. FEBS Lett. 254, 174–176 (1989).

    Article  CAS  Google Scholar 

  25. Levy, N.J., Ramesh, N., Cicardi, M., Harrison, R.A. & Davis, A.E. Type II hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the C1 inhibitor gene. Proc. natn. Acad. Sci. USA 87, 265–268 (1990).

    Article  CAS  Google Scholar 

  26. Baumann, U. et al. Crystal structure of cleaved human α1-antichymotrypsin at 2.7 Å resolution and its comparison with other serpins. J. molec. Biol. 218, 595–606 (1991).

    Article  CAS  Google Scholar 

  27. Stein, P.E. et al. Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature 347, 99–102 (1990).

    Article  CAS  Google Scholar 

  28. Schulze, A.J. et al. Structural transition of α1-antitrypsin by a peptide sequentially similar to β-strand s4A. Eur. J. Biochem. 194, 51–56 (1990).

    Article  CAS  Google Scholar 

  29. Bjork, I., Ylinenjarvi, K., Olson, S.T. & Bock, P.E. Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor. J. biol. Chem. 267, 1976–1982 (1992).

    CAS  PubMed  Google Scholar 

  30. Schreuder, H.A. et al. The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions. Nature struct. Biol. 1, 48–54 (1994).

    Article  CAS  Google Scholar 

  31. Carrell, R.W., Stein, P.E., Fermi, G. & Wardell, M.R. Biological implications of a 3 Å structure of dimeric antithrombin. Structure 2, 257–270 (1994).

    Article  CAS  Google Scholar 

  32. Wei, A., Rubin, H., Cooperman, B.S. & Christianson, D.W. Crystal structure of an uncleaved serpin reveals the conformation of an inhibitory reactive loop. Nature struct. Biol. 1, 251–258 (1994).

    Article  CAS  Google Scholar 

  33. Aertgeerts, K., De Bondt, H.L., De Ranter, C. & Declerck, P.J. Crystallization and X-ray diffraction data of the cleaved form of plasminogen activator inhibitor-1. Proteins 23, 118–121 (1995).

    Article  CAS  Google Scholar 

  34. Holmes, W.E. et al. α2-antiplasmin Enschede: Ala insertion and abolition of plasmin inhibitory activity. Science 238, 209–211 (1987).

    Article  CAS  Google Scholar 

  35. Lawrence, D.A., Olson, S.T., Palaniappan, S. & Ginsburg, D. Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. J. biol. Chem. 269, 27657–27662 (1994).

    CAS  PubMed  Google Scholar 

  36. Engh, R.A., Schulze, A.J., Huber, R. & Bode, W. Serpin Structures. Behring Inst. Mitt. 93, 41–62 (1993).

    CAS  Google Scholar 

  37. Aertgeerts, K., De Bondt, H.L., De Ranter, C. & Declerck, P.J. A model of the reactive form of plasminogen activator inhibitor-1. J. struct. Biol. 113, 239–245 (1994).

    Article  CAS  Google Scholar 

  38. Sancho, E., Tonge, D.W., Hockney, R.C. & Booth, N.A. Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli. Eur. J. Biochem. 224, 125–134 (1994).

    Article  CAS  Google Scholar 

  39. Lomas, D.A., Elliott, P.R., Chang, W.-S.W., Wardell, M.R. & Carrell, R.W. Preparation and characterization of latent α1-antitrypsin. J. biol. Chem. 270, 5282–5288 (1995).

    Article  CAS  Google Scholar 

  40. Wright, H.T. & Blajchman, M., A Proteolytically cleaved mutant antithrombin-Hamilton has high stability to denaturation characteristics of wild type inhibitor serpins. FEBS lett. 348, 14–16 (1994).

    Article  CAS  Google Scholar 

  41. Sancho, E., Declerck, P.J., Price, N.C., Kelly, S.M. & Booth, N.A. Conformational studies on plasminogen activator inhibitor-1 (PAI-1) in active, latent, substrate and cleaved forms. Biochemistry 34, 1064–1069 (1995).

    Article  CAS  Google Scholar 

  42. Gettins, P., Patston, P.A. & Schapira, M. The role of conformational change in serpin structure and function. BioEssays 15, 461–467 (1993).

    Article  CAS  Google Scholar 

  43. Hood, D.B., Huntington, J.A. & Gettins, P.G.W. α1-Proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways. Biochemistry 33, 8538–8547 (1994).

    Article  CAS  Google Scholar 

  44. Olson, S.T., Stephens, A.W., Hirs, C.H.W., Bock, P.E. & Björk, I. Kinetic characterization of the proteinase binding defect in a reactive site variant of the serpin, antithrombin. J. biol. Chem. 270, 9717–9724 (1995).

    Article  CAS  Google Scholar 

  45. Brünger, A.T. X-PLOR version 3.1.a system for X-ray crystallography and NMR. (Yale University Press, New Haven, CT; 1992).

  46. Brünger, A.T. Extension of molecular replacement: a new search strategy based on patterson correlation refinement. Acta Crystallogr. A46, 46–57 (1990).

    Article  Google Scholar 

  47. Jones, T.A., Zou, J.-Y., Cavan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119 (1991).

    Article  CAS  Google Scholar 

  48. Brünger, A.T., Kuriyan, J. & Karplus, M., Crystallographic R factor refinement by molecular dynamics. Science 235, 458–460 (1987).

    Article  Google Scholar 

  49. Read, R.J. Improved fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A42, 140–149 (1986).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Laboratory for Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, E. Van Evenstraat 4, B-3000, Leuven, Belgium

    Kathleen Aertgeerts, Hendrik L. De Bondt & Camiel J. De Ranter

  2. Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, E. Van Evenstraat 4, B-3000, Leuven, Belgium

    Paul J. Declerck

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  1. Kathleen Aertgeerts
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  4. Paul J. Declerck
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Aertgeerts, K., De Bondt, H., De Ranter, C. et al. Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1. Nat Struct Mol Biol 2, 891–897 (1995). https://doi.org/10.1038/nsb1095-891

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