Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg2+ (50mM Mg2+/5mM 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+ (2.5mM Mg2+/5mM Ca2+) as well as under high Ca2+ (5mM Mg2+/45mM 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.
Journal of Molecular Biology – Elsevier
Published: Jun 12, 2013
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