Molecular Basis of Membrane Binding and Activation of Coagulation Factors

项目摘要 对凝血级联反应的分子理解是更有效地解决公共卫生负担的关键 以血栓性疾病和血友病为代表。血栓形成是发病率和死亡率的主要原因，响应- 全球每年约有1000万人死亡，⇠每年有200万起静脉血栓栓塞症事件 仅美国一国。细胞膜是凝血级联反应的中心，因为它为几乎所有 凝血反应。SPECIfic阴离子磷脂通过原子水平对级联反应起着复杂的调节作用。 与凝血因子膜锚的相互作用。密切相关的膜锚显示出明显的不同- TiAl结合和fi城市对阴离子脂类的特异性，由于缺乏详细的解释，现象尚未得到充分解释 结构信息。关于膜结合的原子级结构信息也很匮乏。 由于凝血的膜依赖性，对刺激血栓形成至关重要的大分子复合体 复杂的队形。本申请的目的是阐明阴离子磷脂物种fiCity的关键作用。 和膜结合fi在凝血调节中的作用(目标1)，并建立fi第一个完整的结构模型 一种三元凝血复合体，整合了所有可用的实验信息，并起到了膜的作用 考虑到(目标2)。在目标1中，GLA结构域的膜结合模型，即维生素常见的膜锚 K依赖的凝血因子，将利用加速膜在感兴趣的脂质组合物中开发 表示捕捉自发的膜结合并实现蛋白质-脂质相互作用的增强采样。 特兹。然后将执行高级自由能计算来确定Gla结构域膜与fi的结合。 在目标2中，将首先开发外源三元复合体的蛋白质-蛋白质对接结构，包括所有有益的fi-蛋白质对接结构。 可靠的实验信息。然后将使用这些初始近似结构来确定集合变量， 或复杂形成程度的度量，在非平衡(有偏)的分子动力学中沿其施加力 三元络合物形成的模拟。非平衡模拟将在磷脂双层上进行 以充分考虑膜相互作用的影响。AIMS 1的计算研究结果 和2将用于确定关键的蛋白质-蛋白质和蛋白质-脂相互作用，这些相互作用将进一步 使用实验性的突变研究进行了检测。通过这项工作获得的知识有可能使 靶向特定fiCITY的新疗法的发展，例如针对特定fic-GLA结构域的血栓抑制药 以及治疗血友病的效力增加的重组突变凝血因子。

Project Summary A molecular understanding of the coagulation cascade is key to more effectively addressing the public health burden represented by thrombotic disorders and hemophilia. Thrombosis is a leading cause of morbidity and mortality, respon- sible for approximately 10 million deaths per year worldwide and ⇠2 million venous thromboembolism events per year in the United States alone. The cellular membrane is central to the clotting cascade, as it provides a platform for nearly all coagulation reactions. Specific anionic phospholipids play a complex role in regulating the cascade through atomic-level interactions with coagulation factor membrane anchors. Closely related membrane anchors show markedly differen- tial binding and specificity to anionic lipids, phenomena yet to be adequately explained owing to a lack of detailed structural information. There is also a dearth of atomic-level structural information regarding the membrane-bound macromolecular complexes vital to spurring clot formation as a result of the membrane dependence of coagulation complex formation. The objectives of this application are to elucidate the pivotal role of anionic phospholipid specificity and membrane binding affinity in regulation of coagulation (Aim 1), and to develop the first complete structural model of a ternary coagulation complex integrating all available experimental information and taking the role of the membrane into account (Aim 2). In Aim 1, membrane-bound models of GLA domains, membrane anchors common to vitamin K-dependent coagulation factors, will be developed in lipid compositions of interest using an accelerated membrane representation to capture spontaneous membrane binding and to achieve enhance sampling of protein-lipid interac- tions. Advanced free energy calculations will then be performed to determine GLA domain membrane binding affinity. In Aim 2, protein-protein docked structures of the extrinsic ternary complex will first be developed incorporating all avail- able experimental information. These initial approximate structures will then be used to determine collective variables, or measures of degree of complex formation, along which to apply force in nonequilibrium (biased) molecular dynamics simulations of ternary complex formation. The nonequilibrium simulations will be performed on a phospholipid bilayer to fully take into account the effects of membrane interactions. The results of the computational studies in Aims 1 and 2 will be used to identify key protein-protein and protein-lipid interactions and these interactions will be further examined using experimental mutagenesis studies. The knowledge gained through this work has potential to allow development of novel therapies with targeted specificity, such as thrombotic inhibitors targeting specific GLA domains and recombinant mutant coagulation factors of increased potency for treating hemophilia.