title: Binding of calcium to individual gamma-carboxyglutamic acid residues of human protein C.
abstract: Selectively labeled polypeptides comprising the gamma-carboxyglutamic acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
authors.forename: T L
authors.initials: TL
authors.lastname: Colpitts
authors.forename: M
authors.initials: M
authors.lastname: Prorok
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D011486:Protein C; D015055:1-Carboxyglutamic Acid; D002118:Calcium