Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P-1 residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P-1 Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P-6) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P-1) with Asp- 735 in plasmin, and Arg-29 (P-5') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P-2'-P-4') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P-6-P-5' region of KD1, mutation of a highly conserved Phe at the P-18' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P-1 residue, the nature of other residues flanking the P-1 residue, particularly at P-6 and P-5', strongly influences the inhibitory activity and specificity of human TFPI-2.