Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa.
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.
Dickinson
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Department of Immunology, Scripps Research Institute, La Jolla, CA 92037, USA.
Kelly
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CR
Ruf
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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*
D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa