Abstract
Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3′ Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA–PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3′ interact with PCI rather than uPA, consistent with P3′ variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.
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REFERENCES
Bachmann, F. (1987). Plasminogen Activators in “Hemostasis and Thrombosis: Basic Principles and Clinical Practice” (Eds., Colman, R. W., Hirsh, J., Marder, V. J., Salzman, E. W.) J. B. Lippincott, Philadelphia, pp. 318–339.
Björquist, P., Brohlin, M., Ehnebom, J., Ericcson, M., Kristianson, C., Pohl, G., and Deinum, J. (1994). Biochim. Biophys. Acta. 1209, 191–202.
Bode, W., Turk, D., and Karshikov, A. (1992). Protein Sci. 1, 426–471.
Cooper, S. T., and Church, F. C. (1995). Biochim. Biophys. Acta 1246, 29–33.
Cooper, S. T., Whinna, H. C., Jackson, T. P., Boyd, J. M., and Church, F. C. (1995). Biochemistry 34, 12991–12997.
Crowley, C. W., Cohen, R. L., Lucas, B. K., Lui, G., Shuman, M. A., and Levinson, A. D. (1993). Proc. Natl. Acad. Sci. USA 90, 5021–5025.
DeClerck, Y. A., and Imren, S. (1994). Eur. J. Cancer 30A, 2170–2180.
De Vries, T. J., Quax, P. H. A., Denjin, M., Verrijp, K. N., Verheijen, J. H., Verspaget, H. W., Weidle, U., H., Ruiter, D. J., and van Muijen, G. N. P. (1994). Am. J. Pathol. 144, 70–81.
Espana, F., Estelles, A., Fernandez, P. J., Gilabert, J., Sanchez-Cuenca, J., and Griffin, J. H. (1993). Thromb. Haemost. 70, 989–994.
Fujinaga, M., Sielecki, A. R., Read, R. J., Ardelt, W., Laskowski, M., and James, M. N. G. (1987). J. Mol. Biol. 195, 397–418.
Huang, K., Strynadka, N. C., Bernard, V. D., Peanasky, R. J., and James, M. N. G. (1994). Structure 2, 679–689.
Heeb, M. J., Espana, F., Geiger, M., Collen, D., Stump, D. C., and Griffin, J. H. (1987). J. Biol. Chem. 262, 15813–15816.
Huber, R., and Carrell, R. W. (1989). Biochemistry 28, 8951–8966.
Jackson, S. E., and Fersht, A. R. (1994). Biochemistry 33, 12880–12887.
Janin, J., and Chothia, C. (1976). J. Mol. Biol. 100, 197–211.
Kobayashi, H., Gotoh, J., Shinohara, H., Moniwa, N., and Terao, T. (1994). Thromb. Haemost. 71, 474–80.
Le Bonniec, B. C., Guinto, E. R., and Esmon, C. T. (1992). J. Biol. Chem. 267, 19341–19348.
Le Bonniec, B. C., Guinto, E. R., MacGillivray, R. T. A., Stone, S. R., and Esmon, C. T. (1993). J. Biol. Chem. 268, 19055–19061.
Madison, E. L., Goldsmith, E. J., Gerard, R. D., Gething, M. H., Sambrook, J. F., and Bassel-Duby, R. S. (1990a). Proc. Natl. Acad. Sci. USA 87, 3530–3533.
Madison, E. L., Goldsmith, E. J., Gething, M. J., Sambrook, J. F., and Gerard, R. D. (1990b). J. Biol. Chem. 265, 21423–21426.
Marquart, M., Walter, J., Deisenhofer, J., Bode, W., and Huber, R. (1983). Acta Crystallog. 39B, 408.
Neese, L. L., Cooper, S. T., Jackson, T. P., DiCuccio, M. N., Hoffman, M., and Church, F. C. (1996). FASEB J. 10, A1272 (Abstr. #1578).
Pannekoek, H. (1993). Biomed. Prog. 6, 29–31.
Phillips, J. E., Cooper, S. T., Potter, E. E., and Church, F. C. (1994). J. Biol. Chem. 269, 16696–16700.
Pratt, C. W., and Church, F. C. (1993). Blood Coag. Fibrinol. 4, 479–490.
Pratt, C. W., Macik, B. G., and Church, F. C. (1989). Thromb. Res. 53, 595–602.
Pratt, C. W., Whinna, H. C., and Church, F. C. (1992). J. Biol. Chem. 267, 8795–8801.
Rijken, D. C., and Groeneveld, E. (1991). Biochem. Biophys. Res. Commun. 174, 432–438.
Schechter, I., and Berger, A. (1967). Biochem. Biophys. Res. Commun. 27, 157–162.
Schmitt, M., Janicke, F., Moniwa, N., Chucholowski, N., Pache, L., and Graeff, H. (1992). Biol. Chem. Hoppe-Seyler 373, 611–622.
Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992). J. Biol. Chem. 267, 7588–7595.
Stahl, A., and Mueller, B. M. (1994). Cancer Res. 54, 3066–3071.
Strassburger, W., Wollmer, A., Pitts, J. E., Glover, I. D., Tickle, I. J., Blundell, T. L., Steffens, G. J., Gunzler, W. A., Otting, F., and Flohe, L. (1983). FEBS 157, 219–224.
Stump, D. C., Thienpont, M., and Collen, D. (1986). J. Biol. Chem. 261, 12759–12766.
Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H., and Blow, D. M. (1974). Biochemistry 13, 4212–4228.
Testa, J. E., and Quigley, J. P. (1990). Cancer Metast. Rev. 9, 353–367.
York, J., Li, P., and Gardell, S. (1991). J. Biol. Chem. 266, 8495–8500.
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Jackson, T.P., Cooper, S.T. & Church, F.C. Assessment of the Interaction Between Urokinase and Reactive Site Mutants of Protein C Inhibitor. J Protein Chem 16, 819–828 (1997). https://doi.org/10.1023/A:1026324102618
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DOI: https://doi.org/10.1023/A:1026324102618