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The function of the rigidly conserved amino acid residue R15 in the Ca2+/phospholipid-dependent properties of the gamma-carboxyglutamic acid (Gla)-containing domain (GD) of human Protein C (PC) were investigated through site-directed mutagenesis strategies. A series of recombinant (r) mutants, namely r-[R15K]PC, r-[R15H]PC, r-[R15L]PC, and r-[R15W]PC, were constructed, expressed and purified, and their relevant properties investigated. As revealed by intrinsic fluorescence analysis, all of the variant proteins underwent Ca2+-dependent structural transitions. Nonetheless, they displayed altered binding properties to acidic phospholipid vesicles, and also did not interact with a monoclonal antibody specific for the type of Ca2+-dependent conformation of the GD that characterizes the wild-type protein. On conversion into their activated forms, these variant enzymes possessed less than 10% of the ex vivo plasma anticoagulant activity of wild-type r-PC. Similar activities were found when the r-active PC mutants were assayed directly for inactivation of factor Va and factor VIII, in the complete prothrombinase and tenase complexes respectively. We conclude that R15 is a critical residue in allowing the GD of PC, and probably of other proteins of this class, to adopt a Ca2+-dependent conformation that allows functional phospholipid binding, thus explaining the strict conservation of this amino acid residue in GD modules of various proteins. As a result of an analysis of structural models of the Ca2+-GD complex of PC, it is postulated that hydrogen bonds between the side chain of R15 and the functionally important Gla16 residue, as well as between the side chain of R15 and the carbonyl oxygen in the peptide bond of H10, are critical for adoption of a Ca2+-dependent conformation of the GD that allows functional phospholipid binding.

biochemical journal

Highly conserved residue arginine-15 is required for the Ca2+-dependent properties of the γ-carboxyglutamic acid domain of human anticoagulation Protein C and activated Protein C