Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X.
Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa; however, with the exception of a basic recognition site for the Gla domain of fX, no other interactive site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues Asn-199, Arg-200, and Lys-201 is located at a proper height on the membrane surface to interact with either the C-terminus of the Gla domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wild-type fX and its two mutants which lack either the Gla domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX, was impaired approximately 3-fold. Further kinetic analysis revealed that kcat values with both TF mutants are impaired with no change in Km. These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.
Manithody
Chandrashekhara
C
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Yang
Likui
L
Rezaie
Alireza R
AR
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000737:chemistry; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004815:Epidermal Growth Factor; D001216:Asparagine; D005170:Factor X; D013925:Thromboplastin; D001120:Arginine; D015942:Factor VIIa; D008239:Lysine; D000409:Alanine