title: Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa.
abstract: Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF-VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF-VIIa with macromolecular substrate.
authors.affiliation: Department of Immunology, Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Dickinson
authors.forename: C R
authors.initials: CR
authors.lastname: Kelly
authors.forename: W
authors.initials: W
authors.lastname: Ruf
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015942:Factor VIIa / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa