title: Structural basis of thrombin-mediated factor V activation: the Glu666-Glu672 sequence is critical for processing at the heavy chain-B domain junction.
abstract: Thrombin-catalyzed activation of coagulation factor V (FV) is an essential positive feedback reaction within the blood clotting system. Efficient processing at the N- (Arg(709)-Ser(710)) and C-terminal activation cleavage sites (Arg(1545)-Ser(1546)) requires initial substrate interactions with 2 clusters of positively charged residues on the proteinase surface, exosites I and II. We addressed the mechanism of activation of human factor V (FV) using peptides that cover the entire acidic regions preceding these cleavage sites, FV (657-709)/ (FVa2) and FV(1481-1545)/(FVa3). FVa2 appears to interact mostly with exosite I, while both exosites are involved in interactions with the C-terminal linker. The 1.7-Å crystal structure of irreversibly inhibited thrombin bound to FVa2 unambiguously reveals docking of FV residues Glu(666)-Glu(672) to exosite I. These findings were confirmed in a second, medium-resolution structure of FVa2 bound to the benzamidine-inhibited proteinase. Our results suggest that the acidic A2-B domain linker is involved in major interactions with thrombin during cofactor activation, with its more N-terminal hirudin-like sequence playing a critical role. Modeling experiments indicate that FVa2, and likely also FVa3, wrap around thrombin in productive thrombin·FV complexes that cover a large surface of the activator to engage the active site.
authors.affiliation: Institute for Biomedical Research, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
authors.forename: María Ángeles
authors.initials: MÁ
authors.lastname: Corral-Rodríguez
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
authors.forename: Erick
authors.initials: E
authors.lastname: Hernández-Carvajal
authors.forename: Ricardo
authors.initials: R
authors.lastname: Gutiérrez-Gallego
authors.forename: Pablo
authors.initials: P
authors.lastname: Fuentes-Prior
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry / Q000494:pharmacology; D001550:Benzamidines / Q000737:chemistry / Q000494:pharmacology; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D005165:Factor V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000991:Antithrombins; D001550:Benzamidines; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015943:Factor Va; D005165:Factor V; D013917:Thrombin; C032157:benzamidine; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone