BINDING OF CALCIUM TO INDIVIDUAL GAMMA-CARBOXYGLUTAMIC ACID RESIDUES OF HUMAN PROTEIN-C

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 [C-13]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [C-13] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [C-13] 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 Ca2+- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.