COMPUTATIONAL STRUCTURAL CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS IN HU

HU 中蛋白质-蛋白质相互作用的计算结构表征

• 批准号: 7601529
• 负责人: DIVI VENKATESWARLU
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601529
• 关键词: Address; Binding; Blood Clot; Blood coagulation; Coagulation Process; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Data; Databases; Disease; Endopeptidases; Engineering; Enzyme Precursors; Enzymes; Event; Factor VIIIa; Factor X; Failure; Funding; Gene Mutation; Goals; Grant; Hemophilia A; Hemorrhage; Homology Modeling; Human; Individual; Institution; Laboratories; Medical center; Methods; Modeling; Molecular; Molecular Conformation; Pathway interactions; Patients; Peptide Hydrolases; Phase; Play; Production; Protein Interaction Mapping; Protein-Protein Interaction Map; Proteins; Proteolysis; Rate; Reaction; Research; Research Personnel; Resources; Roentgen Rays; Role; Series; Serine Protease; Site; Site-Directed Mutagenesis; Solutions; Source; Stream; Structural Models; Structure; Techniques; Tertiary Protein Structure; Thrombin; United States National Institutes of Health; aqueous; base; cancer procoagulant; cofactor; comparative; enzyme substrate; inhibitor/antagonist; interest; molecular dynamics; professor; protein protein interaction; research study; therapeutic protein

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 具体目标 该提案的目标是通过采用先进的计算机模拟方法来了解内在凝血途径中酶-底物-辅因子相互作用的结构基础。在拟议的资助期内，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。内在途径涉及酶原（称为酶原、丝氨酸蛋白酶因子 X）通过蛋白酶 IXa 的蛋白水解激活。该反应具有催化惰性，并且在 FIXa 与辅因子 FVIIIa 结合之前不会以具有生物学意义的速率发生。复合物 FIXa:FVIIIa 被称为内在 Xase 复合物，在激活酶原因子 X 从而启动凝血级联的一系列下游事件中发挥关键作用。基因突变或获得性抑制剂导致的 FVIII 水平故障会导致轻度至重度出血性疾病，称为血友病 A。无法形成活性 Xnase 复合物会导致 FXa 产生急剧减少，而 FXa 是凝血酶爆炸性形成和相应的下游凝血事件所必需的。因此，人们对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非常感兴趣。我们建议采用计算蛋白质建模和水相分子动力学方法来开发基于部分 X 射线晶体结构数据和比较同源建模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 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）。 （目标 III）构建因子 FVIIIa、FIXa 和酶原因子 FX 之间的三元复合物，以描绘 FX 激活过程中的蛋白质识别位点。 （目标 IV）开发酶 FIXa 和酶原 FX 之间的二元复合物，以了解 FIXa:FX 的辅因子独立关联。通过提供结构理解，我们希望解决以下问题：i) 各个​​蛋白质（即 FVIIIa 和 FIXa）的哪些特定域参与内在 Xnase 复合物的形成？ ii) 辅因子结合时的域间相互作用和构象有何不同？ iii) 内源途径中酶原 FX 蛋白水解的激活机制是什么？ 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，如果可以，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？预测的分子内和分子间水平的蛋白质-蛋白质相互作用数据将通过罗彻斯特医学中心 Philip J. Fays 教授实验室的定点诱变实验进行验证。