Investigation of protein interactions in intrinsic blood coagulation pathway

我们建议使用计算蛋白质模拟和水相分子动力学方法来开发基于部分X射线晶体结构数据和比较同源模拟技术的三维溶液结构模型。在所要求的资助期内，我们建议开发与内在凝血途径相关的多域蛋白的结构组装。该项目的具体目标是：(目标一)：开发因子VIIIa、IXa和Fx的动态平衡结构模型(目标二)，以模拟辅助因子FVIIIa和因子FixA之间的二元复合体(也称为内在Xnase复合体)。(目的III)构建因子FVIIIa、FIXA和酵素原因子Fx的三元复合体，以确定Fx激活过程中的蛋白质识别位点。(目的IV)开发酶FIXA和酶原FX之间的二元复合体以了解FixA：Fx之间的辅因子独立关联通过提供结构上的理解，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，参与内在Xnase复合体的形成？Ii)共因子结合时，结构域-结构域的相互作用和构象有多大不同？Iii)内在途径中酶原FX蛋白分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，是否可以使用原子细节上的蛋白质-蛋白质相互作用数据来设计治疗性蛋白质？PHS 398/2590(09/04版)第9页延续格式页首席调查员/计划总监(最后、第一、中间)：Venkateswarlu，Divi

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? PHS 398/2590 (Rev. 09/04) Page 9 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Venkateswarlu, Divi