COMPUTATIONAL STRUCTURAL CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS IN HU

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 具体目标本提案的目标是通过采用先进的计算机模拟方法，了解内源性凝血途径中酶-底物-辅因子相互作用的结构基础。在建议的资助期内，我们计划开发与凝血级联内在途径相关的人类形式的酶（FIXa）、底物（FX）和辅因子（FVIIIa）蛋白之间的蛋白质相互作用位点的完整分子图谱。内源性途径涉及蛋白酶IXa对酶原（称为酶原，丝氨酸蛋白酶因子X）的蛋白水解激活。该反应具有催化惰性，在FIXa与辅因子FVIIIa结合之前不会以生物学显著速率发生。FIXa：FVIIIa复合物被称为内源性Xase复合物，在激活酶原因子X并由此引发一系列凝血级联的下游事件中起关键作用。基因突变或获得性抑制剂导致的FVIII水平的功能障碍可导致轻度至重度出血性疾病，称为血友病A。未能形成活性Xnase复合物导致FXa产生的急剧减少，这是凝血酶爆发性形成和相应的血液凝固下游事件所需的。因此，阐明单个蛋白质和相关蛋白质-蛋白质复合物的确切结构和构象具有极大的兴趣。我们建议采用计算蛋白质建模和水相分子动力学方法开发三维溶液结构模型的基础上部分X-射线晶体结构数据和比较同源建模技术。在申请资助期间，我们建议发展与内在凝血途径相关的多结构域蛋白质的结构组装。该项目的具体目标是：（目标I）：开发因子VIIIa、IXa和FX的动态平衡结构模型（目标II），以模拟辅因子FVIIIa和因子FIXa之间的二元复合物（也称为内在Xnase复合物）。(Aim III）构建因子FVIIIa、FIXa和酶原因子FX之间的三元复合物，以试图描绘FX活化过程中的蛋白质识别位点。(Aim IV）开发酶FIXa和酶原FX之间的二元复合物，以理解FIXa：FX的辅因子独立缔合通过提供结构理解，我们希望解决以下问题：i）单个蛋白质的哪些特定结构域，即，FVIIIa和FIXa是否参与了内源性Xnase复合物的形成？ii）在辅因子结合时，结构域-结构域相互作用和构象有多大不同？iii）内源性途径中酶原FX蛋白水解的激活机制是什么？以及iv）我们能否破译大量血友病A和B患者突变数据库与所提出的结构模型之间的结构-功能相关性，如果是的话，我们能否在原子细节上使用蛋白质-蛋白质相互作用数据来设计治疗性蛋白质？预测的蛋白质-蛋白质相互作用数据，在分子内和分子间水平将通过在罗切斯特医学中心的Philip J. Fays教授实验室的位点特异性诱变实验进行验证。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SPECIFIC AIMS The goal of this proposal is to understand the structural basis of the enzyme-substrate-cofactor interactions in the intrinsic blood coagulation pathway by employing advanced computer simulation methods. In the proposed funding period, we plan to develop a complete molecular mapping of protein interaction sites among the human forms of enzyme (FIXa), substrate (FX) and co-factor (FVIIIa) proteins associated with intrinsic pathway of coagulation cascade. Intrinsic pathway involves the proteolytic activation of pro-enzyme, known as zymogen, serine-protease factor X by protease enzyme IXa. This reaction is catalytically inert and does not occur at biologically significant rate until FIXa binds to co-factor FVIIIa. The complex FIXa:FVIIIa, known as intrinsic Xase complex, plays key role in activating zymogen factor X and thereby initiating a series of down-stream events of coagulation cascade. Malfunction at the level of FVIII by either genetic mutations or acquired inhibitors causes a mild to severe bleeding disorder called hemophilia A. Failure to form an active Xnase complex results in a dramatic reduction of FXa production, which is required for the explosive formation of thrombin and corresponding downstream events of blood clotting. Therefore, there is a great interest in elucidating the exact structures and conformations of the individual proteins and the related protein-protein complexes. We propose to employ computational protein modeling and aqueous-phase molecular dynamics methods to develop three-dimensional solution structural models based on partial X-ray crystal structural data and comparative homology modeling techniques. During the requested funding period, we propose to develop structural assembly of multi-domain proteins associated with intrinsic blood coagulation pathway. The specific objectives of the project are: (Aim I): to develop the dynamically equilibrated structural models for factors VIIIa, IXa and FX (Aim II) to model the binary complex between co-factor FVIIIa and factor FIXa (also known as intrinsic Xnase complex). (Aim III)to build the ternary complex among factors FVIIIa, FIXa and zymogen factor FX in an effort to delineate the protein recognition sites during FX activation. (Aim IV) to develop the binary complex between enzyme FIXa and zymogen FX to understand co-factor independent association of FIXa:FX By providing a structural understanding, we hope to address the following questions: i) What specific domains of the individual proteins, i.e., FVIIIa and FIXa, are involved in the intrinsic Xnase complex formation? ii) How different are the domain-domain interactions and conformations upon co-factor binding? iii) What is the activation mechanism for zymogen FX proteolysis in intrinsic pathway? And iv) Can we decipher the structure-function correlation between the vast amount of hemophilia A and B patient mutational database and the proposed structural models, and if yes, can one use the protein-protein interaction data at the atomic details to engineer the therapeutic proteins?. The predicted protein-protein interaction data, at both intra and inter-molecular level would be validated by site-specific mutagenesis experiments in Professor Philip J. Fays laboratory at Rochester Medical Center.