Previous studies from this laboratory have been directed toward elucidation of the roles of individual gamma-carboxyglutamic acid (Gla) residues in Gla domain-related Ca2+-directed properties of human protein C (PC) and activated protein C (APC). On the basis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla(6), Gla(14), and Gla(19) may not be essential for properties associated with the Ca2+-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated that radical mutations to Val of Gla residues 14 and 19 resulted in 94% and 82%, respectively, of the Gla domain-related, Ca2+- and phospholipid- (PL-) dependent anticoagulant (APTT) activity of wild-type recombinant (wtr) APC, while [Gla(6)-->Val]r-APC showed a complete loss of this same activity. The more conservative mutant [Gla(6)-->Gln]r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla(6) -->Asp]r-APC was nearly fully active. As with wtr-PC, both [Gla(6)-->Val]r-PC and [Gla6-->Gln]r-PC displayed Ca2+ dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca2+-induced conformation. However, Ca2+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca2+-mediated binding parameters of [Gla(6)-->Val]r-PC and [Gla(6)-->Gln]r-PC to acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca2+/bovine prothrombin fragment 1 complex. We conclude that Gla(14) and Gla(19), and the sole Ca2+ (Ca-7) that is coordinated to these two residues, are not essential for the Gla domain-related Ca2+- and PL-dependent anticoagulant properties of PC and APC. A more complex situation exists with Gla(6). The model suggests that only a single carboxylate of this residue is employed in Ca2+ coordination, and this group interacts with two different Ca2+ ions, viz., Ca-4 and Ca-5. The lone carboxylate of Asp may be able to substitute effectively for this same group in Gla(6), thus providing functional activity to [Gla(6)-->Asp]r-APC, and to a much lesser degree Gin may also be able to serve a coordination function. This suggests that Gla(6) is one of the functionally essential Ca2+ binding sites.
