Computer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin

International audienceComputer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin Dear Sir, Tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine proteinase inhibitor that strongly inhibits trypsin, plasmin, factor XIa and plasma kallikrein [1], and is mainly associated with extracellular matrices [2]. By inhibiting plasmin, TFPI-2 thus efficiently decreases activation of several metalloproteinases [2] and reduces tumor invasion and metastasis [3,4]. In addition, TFPI-2 can also regulate plasmin in atherosclerotic plaques by modulating extracel-lular proteolytic mechanisms [5]. TFPI-2, which is a member of the Kunitz-type serine proteinase inhibitor family, consists of three tandemly repeated Kunitz-type domains, the first of which (K1) exhibits similarities in amino acid sequence to TFPI-1 and bovine pancreatic trypsin inhibitor (BPTI) (45% and 40%, respectively). The inhibition of serine proteinases by Kunitz-type inhibitors involves their binding to K1 and P1 residue that interacts with the proteinase pocket previously identified to be an arginine or a lysine residue [6-9]. Using site-directed mutagenesis experiments, Kamei et al. [10] reported that a TFPI-2 mutant with glutamine substituted for arginine lost its inhibitory activity towards trypsin and plasmin. Several structures showing the interaction of one serine proteinase with a Kunitz-type inhibitor, i.e. thrombin with BPTI [6], factor Xa/VIIa with TFPI-1 [7,8] and alpha-chymotrypsin with BPTI [9], have been determined crystallographically. However , TFPI-2 structure and plasmin complexed with a Kunitz-type inhibitor have not to date been described. This report is the first to address the structural basis for molecular recognition of plasmin by the K1 domain of TFPI-2 (TFPI-2/K1). The 3D models of TFPI-2/K1 was built using the automatic comparative modeling server swissPDB (http:// www.expasy.org). The structure of TFPI-1/K2 (44% identity with TFPI-2/K1 in 55 residues) was used as template to determine TFPI-2/K1. The resulting model was further energy-minimized using the steepest descent algorithm (SYBYL 6.9, Tripos, http://www.tripos.com), the atoms of the backbone being constrained during this step, and un-constrained using the conjugate gradient algorithm until the maximum derivative was < 0.1 kcal/mol/A ˚. After checking the stereochemical quality (WHATIF, http://www.cmbi. kun.nl/), all parameters were good for ensemble structure. The TFPI-2/K1 model and plasmin (coordinates extracted from the PDB file 1 bml) were superimposed on the Ca atoms of the TFPI-1/trypsin complex (coordinates extracted from the PDB file 1tfx). Finally, the few atoms that overlapped at the complex interface were relieved by energy minimization. The electrostatic potentials of TFPI-2/K1 and plasmin were calculated with swissPDB viewer 3.7b software and using formal charges at pH 7.4 (arginine, lysine, N-terminus, + 1; glutamate, aspartate and the C-terminus, À 1; and histidine, neutral), ionic strength in the aqueous environment of 0.15 M and dielectric constant of 80. The three-dimensional structure of TFPI-2/K1 exhibited the typical Kunitz-type proteinase inhibitor folding, i.e. a double stranded anti-parallel h-sheet from Arg20 to Phe33, and an a-helix from Trp48 to Ala54. The core of the domain, comprising the secondary structure elements and the three disulfide bonds, was highly conserved. TFPI-2/K1 exhibited the same overall fold as BPTI [6] and TFPI-1/K2 [7], with rms deviations (root-mean-square) for all 55 Ca atom positions of 0.70 and 1.09 A ˚ , respectively. These values are in agreement with the 40% identity with BPTI and 45% identity with TFPI-1/K2. The binding loop of TFPI-2/K1 (Leu19-Tyr33) was in direct contact with the active site of plasmin involving the characteristic main-main conforma-tional and intermolecular hydrogen bond interactions of canonical binding proteinase inhibitors [7-9]. Indeed, as shown in Table 1, TFPI-2/K1 residues positioned close to the active site of plasmin (PV2, PV1, P1 and P3 residues) were involved in hydrogen bonds. As compared with the TFPI-1/trypsin complex [7], two additional hydrogen bonds were found at the interface between TFPI-2/K1 and plasmin, probably improving the complementarity between the two molecules. The side-chain of TFPI-2 P1 residues (Arg15) extended into the plasmin-specific pocket, with its guanidi-nium group forming a salt bridge with the carboxylate group 0049-3848/$-see front matter