INTERMOLECULAR INTERACTIONS BETWEEN PROTEIN-C INHIBITOR AND COAGULATION PROTEASES

Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr(352) to Arg(357)) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha(1)-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe(353), With 2 and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr(352), With 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu(217) of each protease. Changing Arg(357) at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu(220). Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.