Liaw P C, Fredenburgh J C, Stafford A R, Tulinsky A, Austin R C, Weitz J I
McMaster University and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada.
J Biol Chem. 1998 Apr 10;273(15):8932-9. doi: 10.1074/jbc.273.15.8932.
Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.
共结晶学研究表明，人凝血酶原片段2（F2）与凝血酶的相互作用涉及F2的kringle内环与凝血酶的阴离子结合外位点II之间形成盐桥。当F2与凝血酶结合时，已显示会引起该酶活性位点和外位点I的构象变化。我们使用血浆、重组和合成的F2肽（分别为F2、rF2和sF2）进一步定位了F2上的凝血酶结合结构域。F2、rF2-(1-116)、rF2-(55-116)和sF2-(63-116)，它们都包含kringle内环（64-93位残基）和酸性COOH末端连接肽（94-116位残基），可与凝血酶琼脂糖结合。相比之下，kringle内环类似物sF2-(63-90)或COOH末端连接肽类似物sF2-(92-116)则不结合。因此，与晶体结构预测相反，COOH末端连接肽以及kringle内环都参与了F2与凝血酶的相互作用。F2和sF2-(63-116)分别以4.1和51.3 microM的Kd值与荧光标记的活性位点封闭的凝血酶饱和结合。当78位的缬氨酸被谷氨酸取代时，sF2-(63-116)对凝血酶的亲和力增加约5倍（Kd = 10 microM）。F2和sF2-(63-116)与凝血酶的外位点II结合，因为两者都将抗凝血酶催化的凝血酶抑制速率降低了约4倍。相比之下，只有F2减缓了抗凝血酶对凝血酶的非催化失活速率。与F2一样，sF2-(63-116)会诱导凝血酶活性位点和外位点I的变构变化，因为它分别改变了凝血酶介导的发色底物水解速率，并从活性位点封闭的凝血酶中置换出荧光标记的水蛭素54-65。这两种肽还以浓度依赖的方式延长了纤维蛋白原的凝血酶凝血时间，反映了它们对活性位点和/或外位点I的影响。这些研究进一步深入了解了F2中引起凝血酶功能变化的区域。
