INFLUENCE OF SPECIFIC GAMMA-CARBOXYGLUTAMIC ACID RESIDUES ON THE INTEGRITY OF THE CALCIUM-DEPENDENT CONFORMATION OF HUMAN PROTEIN-C

The concentration of Ca2+ that produced 50% of the saturable intrinsic fluorescence change (C50) of wild-type (wt) recombinant (r) human protein C (PC) was 0.40 mM. The C50 for Ca2+ increased <2.5-fold for the following r-PC variants (Gla is gamma-carboxyglutamic acid): [Gla6 --> Asp]r-PC, [Gla7 --> Asp]r-PC, [Gla14 --> Asp]r-PC, [Gla19 --> Asp]r-PC, or [Gla25 --> Asp]r-PC, and approximately 4-6-fold for [Gla20 --> Asp]r-PC and [Gla29 --> Asp]r-PC. Much more dramatic increases in the C50 for Ca2+ were observed for [Gla16 --> Asp]r-PC (>75-fold) and [Gla26 --> Asp]r-PC (ca. 30-fold). A substantially larger maximum fluorescence change (>3-fold) as compared to that for wtr-PC, was also found in the case of the Ca2+/[Gla16 --> Asp]r-PC complex, suggesting that the final Ca2+-induced conformation for this variant is dissimilar to that for wtr-PC and the above mutants. When a mutation was constructed at Arg15 ([Arg15 --> Leu]r-PC), a residue conserved in all Gla-containing coagulation proteins, no fluorescence alteration occurred upon addition of Ca2+. The C50 for Ca2+ for promotion of the binding of the Ca2+-dependent, Gla-domain-directed, conformational monoclonal antibodies, JTC-1 and JTC-3, to wtr-PC was 3.0 and 4.0 mM, respectively. A similar C50 value was found for [Gla6 --> Asp]r-PC. In the case of each antibody, approximately 4-6-fold higher C50 values for Ca2+ were found for the mutants; [Gla14 --> Asp]r-PC, [Gla19 --> Asp]r-PC, and [Gla29 --> Asp]r-PC. Ca2+ did not promote binding of either of these antibodies to the following variants; [Gla6 --> Asp]r-PC, [Gla7 --> Asp] r-PC, [Arg15 --> Leu]r-PC, [Gla16 --> Asp]r-PC, [Gla20 --> Asp]r-PC, and [Gla26 --> Asp]r-PC. The results of this study suggest that adoption of the Ca2+-dependent conformation of PC is greatly dependent upon the presence of specific essential Gla residues, particularly those, namely Gla16 and Gla26, shown in the crystal structure of the prothrombin Gla domain/Ca2+ complex to be involved with coordination of Ca2+ ions not exposed to the surface. Of similar importance is Arg15. On the other hand, Gla residues at positions 14 and 19 are much less important in directing this same conformation. This finding is readily reconciled with the above crystal structure, which shows that these latter 2 residues are mainly responsible for coordination of a surface-exposed Ca2+ that is present at the end of the Ca2+-ion channel.