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 Ca2+-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 Ca2+-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. Sr2+ and Ba2+, with ionic radii larger than the ionic radius of Ca2+, can bind to Ca2+-free ACF I (apo-ACF I), while Mg2+, with an ionic radius smaller than that of Ca2+, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca2+, Sr2+, and Ba2+ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr2+-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca2+–ACF I, while Ba2+-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca2+–ACF I. Mg2+ and Sr2+, with ionic radii close to that of Ca2+, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba2+, with a much larger ionic radius than Ca2+, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca2+, Sr2+, and Ba2+ 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 Mg2+, Ca2+, and Sr2+ ions to FXa may be essential for the recognition between FXa and ACF I.