Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg2 + (50 mM Mg2 +/5 mM Ca2 +), have three of seven Ca2 + sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg2 + at positions 1, 4, and 7. We now report structures under low Mg2 + (2.5 mM Mg2 +/5 mM Ca2 +) as well as under high Ca2 + (5 mM Mg2 +/45 mM Ca2 +). Under low Mg2 +, four Ca2 + and three Mg2 + occupy the same positions as in high-Mg2 + structures. Conversely, under low Mg2 +, reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca2 + and positions 1 and 7 by Mg2 +. Nonetheless, in direct binding experiments, Mg2 + replaced three Ca2 + sites in the unliganded Protein C or APC. Further, the high-Ca2 + condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg2 + enhanced phospholipid binding to FVIIa and APC at physiological Ca2 +. Additionally, Mg2 + potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg2 + condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg2 + and Ca2 + act in concert to promote coagulation and anticoagulation.