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.

Characterization of the interaction between the light chain of factor VIIa and tissue factor

“…A conceivable explanation is that Ca 2+ ‐binding sites in the Gla domain play a role in the Ca 2+ ‐dependent fVIIa activity in the presence of sTF, i.e. in sTF binding, considering the broad range of affinities represented within this group of sites [37]. These sites have been hypothesized to be important for the expression of TF‐interactive epitopes [38].…”

Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

“…In contrast, factor VIIa binding to phospholipid and F1 A2 could not be induced by Mgz'. Applying prothrombin and protein C as models for the Gla domain of factor VIIa, presumably Mg" can not replace Ca" in the interior binding sites, corresponding to Ca" ions 2-5 in the Ca2'~ structure of prothrombin fragment 1, and/or replace the solvent-accessible Ca2+ ions (1, 6 and 7 ; Soriano-Garcia et al, 1992), presumably Ca6, required for phospholipid binding (Colpitts et al, 1995). By comparison of the apo and Ca'+ structures of the factor X Gla domain, it was seen that buried Ca2+ ions occupy the space that harbours the membrane-interactive residues Phe4, Leu5 and Val8 in the apo structure and it was suggested that the binding of these Caz+ ions brings about the phospholipid accessibility of those three residues (Sunnerhagen et al, 1995).…”

Determination of the Solution Structures of Conantokin-G and Conantokin-T by CD and NMR Spectroscopy

“…Gla residues were originally identified in the vitamin K-dependent blood-clotting factors including prothrombin. Subsequently they were found in other vertebrate proteins such as osteocalcin (calcium-binding protein) (10,11) and have been implicated in Ca 2ϩ binding (12,13). The discovery of Gla residues in several Conus peptides has established that this posttranslational modification has a much wider phylogenetic distribution than previously thought (14).…”