Xu Xiaolong, Zhang Liyun, Shen Dengke, Wu Hao, Peng Lili, Li Jiehua
Department of Chemistry, University of Science and Technology of China, Hefei, People's Republic of China.
J Biol Inorg Chem. 2009 May;14(4):559-71. doi: 10.1007/s00775-009-0470-3. Epub 2009 Jan 31.
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.
从尖吻蝮蛇毒中分离得到的抗凝因子I（ACF I）是一种活化凝血因子X（FXa）结合蛋白，它以Ca(2+)依赖的方式结合，具有显著的抗凝活性。分别采用等温滴定量热法、荧光法、圆二色性法和表面等离子体共振法研究了碱土金属离子与ACF I结合的热力学，以及碱土金属离子对盐酸胍（GdnHCl）诱导的ACF I解折叠和ACF I与FXa结合的影响。结果表明，占据ACF I中Ca(2+)结合位点的阳离子的离子半径对ACF I与碱土金属离子的结合亲和力以及ACF I对GdnHCl变性的结构稳定性有至关重要的影响。离子半径大于Ca(2+)的Sr(2+)和Ba(2+)可以与无Ca(2+)的ACF I（脱辅基ACF I）结合，而离子半径小于Ca(2+)的Mg(2+)对与脱辅基ACF I的结合显示出极低的亲和力。ACF I两个位点中Ca(2+)、Sr(2+)和Ba(2+)离子的所有结合主要是焓驱动的，熵对它们不利。Sr(2+)稳定的ACF I对GdnHCl变性的抗性略低于Ca(2+)-ACF I，而Ba(2+)稳定的ACF I对GdnHCl变性的抗性远低于Ca(2+)-ACF I。离子半径与Ca(2+)接近的Mg(2+)和Sr(2+)可以与FXa结合，因此也能诱导ACF I与FXa结合，而离子半径比Ca(2+)大得多的Ba(2+)不能支持ACF I与FXa结合。我们的观察结果表明，ACF I两个位点中Ca(2+)、Sr(2+)和Ba(2+)离子的结合增加了ACF I的结构稳定性，但这些结合对于ACF I与FXa的结合不是必需的，并且Mg(2+)、Ca(2+)和Sr(2+)离子与FXa的结合可能是FXa与ACF I之间识别的关键。
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