PDF(Pubmed)
Abstract：
Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.
摘要：
止血依赖于丝氨酸蛋白酶及其辅因子的反应网络以形成血凝块。凝血因子IXa（蛋白酶）在止血中起着至关重要的作用，这从与缺乏凝血因子IXa相关的出血疾病中可以明显看出。特异性靶向单个凝血因子的RNA适体具有作为抗凝剂和作为结构与功能之间关系的探针的潜力。这里,我们报道了人因子IXa的X射线结构，该结构没有以apo形式或与对因子IXa具有特异性的抑制性适体复合的形式与活性位点结合的配体。适体与催化结构域中的外位点结合并变构扭曲活性位点。我们的研究揭示了IXa态的构象集合，其中Trp215在活性位点附近的大移动驱动闭合(适体结合)之间的功能转变，潜伏（apo），和开放(底物结合)状态。与其他凝固蛋白酶相比，apo酶的潜伏状态可能会影响IXa独特的差催化活性。exosite,适体与之结合，与VIIIa和肝素的结合有关，两者都调节IXa功能。我们的发现揭示了exosite驱动的IXa功能变构调节的重要性以及重新平衡止血以获得治疗效果的新策略。
