Mg2+ Is Required for Optimal Folding of the γ-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+
Authors: Vadivel Kanagasabai , Agah Sayeh , Messer Amanda S. , Cascio Duilio , Bajaj Madhu S , Krishnaswamy Sriram , Esmon Charles T , Padmanabhan Kaillathe , Bajaj S. Paul 
Published: October 2019

Abstract
Abstract 1172
Equilibrium dialysis experiments indicate that the Gla domain of coagulant protein factor (F) VIIa and of anticoagulant protein C (PC) each bind seven Ca2+ in the absence and four in the presence of physiologic Mg2+ (0.6 mM). The previous x-ray structure of FVIIa/soluble (s) tissue factor (TF) in the presence of 5 mM Ca2+/45 mM Mg2+ (5Ca/45Mg) (Bajaj et al., J Biol Chem, 281, 24873–24888, 2006) revealed that sites 2,3,5 and 6 were occupied by Ca2+ while sites 1, 4 and 7 contained Mg2+ (Tulinsky numbering, Biochemistry 31, 2554–2566, 1992). Since these concentrations of metal ions were supraphysiologic, we sought to obtain crystals under concentrations of Ca2+ and Mg2+ approaching those found in blood. We have solved the structures of FVIIa/sTF under three new conditions: 2.5 mM Ca2+/1.25 mM Mg2+ at 2.8 Å (2.5Ca/1.25Mg); 5 mM Ca2+/2.5 mM Mg2+ at 1.8 Å (5Ca/2.5Mg); and 45 mM Ca2+/5 mM Mg2+ at 1.7 Å (45Ca/5Mg). For 2.5Ca/1.25Mg, the Gla domain of FVIIa was disordered indicating insufficient concentrations of Ca2+/Mg2+ to fold the Gla domain. Interestingly, folding of the Ω-loop for 5Ca/2.5Mg was similar to that reported under 5Ca/45Mg, with identical positions for four Ca2+ and three Mg2+. In contrast, while the folding of the Ω-loop at 45Ca/5Mg was similar to that reported only in the presence of Ca2+ (Banner et al., Nature, 380, 41–46,1996); however, positions 1 and 7 contained Mg2+ (Fig. 1A). Thus, it would appear that four Ca2+ and three Mg2+ ions are bound to the circulating FVII/FVIIa. Moreover, circulating FVII/FVIIa will need high Ca2+ concentrations to have its position four switched from Mg2+ to Ca2+.Fig. 1.A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations.Fig. 1. A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations.
All seven metal sites in the structure of PC-Gla complexed to endothelial protein C receptor (EPCR) had been refined with Ca2+ despite the presence of 5 mM Ca2+ and 5 mM Mg2+ in the crystallization buffer (Oganesyan et al., J Biol Chem, 277, 24851–24854, 2002). Based on our findings with VIIa/sTF, we revisited the PC-Gla/EPCR structure. We determined that positions 1 and 7 are Mg2+ sites while 2, 3, 4, 5 and 6 are Ca2+ sites in the PC-Gla/EPCR structure (Fig. 1B). This contrasts to four Ca2+ and three Mg2+ bound to VIIa/TF at 5Ca/2.5Mg. Since five Ca2+ and two Mg2+ were seen in VIIa/sTF at 45Ca/5Mg, it would appear that position 4 can accommodate either Ca2+ or Mg2+ depending upon conditions. We propose that at near physiologic Ca2+ and Mg2+, ligation of the Gla domain with EPCR and possibly phospholipid (PL) membranes facilitates substitution of the Mg2+ bound at the position four with Ca2+. This metal switch may be essential to achieve a favorable conformation of the Gla domain Ω-loop for optimal ligand of PL binding.
Mg2+ also enhanced the Ca2+-dependent interaction of FVIIa or activated protein C (APC) to PL assessed by surface plasmon resonance. Binding of FVIIa and APC each was saturated by the physiological concentration of Ca2+ (1.1 mM) in the presence of physiological Mg2+ (0.6 mM). In contrast, only half-saturable binding was observed at the physiological concentration of Ca2+ in the absence of Mg2+. Further, 0.6 mM Mg2+ potentiated (∼2.5-fold) the PL-dependent activation of FX by FVIIa/TF at 1.1 mM Ca2+. Similarly, Mg2+ potentiated (∼3-fold) the activation of FX by FVIIa/TF assembled on the endotoxin-stimulated monocyte surface. PL-dependent inactivation of FVa by APC was also enhanced ∼3-fold by 0.6 mM Mg2+ at 1.1 mM Ca2+. At saturating Ca2+ (5 mM), the activation of FX by FVIIa/TF or the inactivation of FVa by APC was similar to that with 1.1 mM Ca2+/0.6 mM Mg2+. Thus Mg2+ at physiologic concentrations augments PL- or natural membrane-dependent coagulation and anticoagulation at the plasma concentration of Ca2+.
We propose that vitamin K-dependent clotting and anti-clotting proteins circulate in blood with four Ca2+ ions bound. The remaining three (or more in FIX and FX) divalent metal binding sites in each Gla domain are occupied by Mg2+. The conformation of the Ω-loop in circulating vitamin K- dependent proteins is not favorable for binding to PL but it is achieved by switching Mg2+ at position four to Ca2+. Thus, the metal ion at position four regulates PL-dependent coagulation and anticoagulation reactions.

Disclosures:
No relevant conflicts of interest to declare.