Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability.

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca(2+)-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca(2+)-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr(2+) and Ba(2+), with ionic radii larger than the ionic radius of Ca(2+), can bind to Ca(2+)-free ACF I (apo-ACF I), while Mg(2+), with an ionic radius smaller than that of Ca(2+), shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr(2+)-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca(2+)-ACF I, while Ba(2+)-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca(2+)-ACF I. Mg(2+) and Sr(2+), with ionic radii close to that of Ca(2+), can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba(2+), with a much larger ionic radius than Ca(2+), cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca(2+), Sr(2+), and Ba(2+) ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg(2+), Ca(2+), and Sr(2+) ions to FXa may be essential for the recognition between FXa and ACF I.