COMPUTATIONAL STRUCTURAL CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS IN HU

点击翻译按钮获取中文摘要

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.