Molecular mechanism of protein C activation

Protein C激活的分子机制

• 批准号: 9918442
• 负责人: Enrico Di Cera
• 金额: $ 37.88万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918442
• 关键词: Active Sites; Affect; Amino Acid Sequence; Anticoagulant therapy; Anticoagulants; Architecture; Binding; Biology; Blood coagulation; Cardiovascular Diseases; Cause of Death; Cessation of life; Chimeric Proteins; Coagulation Process; Complement; Complex; Crystallization; Development; Docking; EGF gene; Engineering; Environment; Enzyme Precursors; Enzymes; Escherichia coli; Excision; Expectancy; Family; Feedback; Fluorescence Resonance Energy Transfer; Generations; Investigation; Isotope Labeling; Kinetics; Knowledge; Life Expectancy; Life Style; Measurement; Molecular; Molecular Conformation; Mutagenesis; Pathway interactions; Peptide Hydrolases; Phase; Physiological; Productivity; Property; Protein C; Protein Conformation; Prothrombin; Reaction; Reagent; Regulation; Research Project Grants; Resolution; Roentgen Rays; Role; Side; Site; Site-Directed Mutagenesis; Structure; Substrate Specificity; Tertiary Protein Structure; Testing; Thrombin; Thrombomodulin; Thromboplastin; Trypsin; Vertebral column; X ray spectroscopy; alpha-Thrombin; cofactor; cysteinyltyrosine; disability; improved; innovation; interest; member; mindfulness; mutant; prethrombins; protein activation; response; single molecule; statistics; stem; success

Abstract The proposed research project focuses on the thrombomodulin-dependent activation of protein C by thrombin as a key regulatory feedback loop of the coagulation response. Our interest in this reaction stems from its physiological relevance, the lack of a molecular understanding of its mechanism and the translational opportunities that might ensue from advances in basic knowledge. Investigation of protein C is motivated by our recent success in the crystallization of prothrombin and characterization of its structure in solution, as well as by new reagents developed in the lab, i.e., a derivative of protein C devoid of the auxiliary Gla and EGF domains (miniPC) expressed in E. coli for isotope labeling and a fusion protein (FP) of thrombin with the EGF456 domains of thrombomodulin that recapitulates the structural and functional properties of the thrombin- thrombomodulin complex. Our guiding hypothesis is that thrombomodulin (the cofactor) functions by optimizing the environment of the catalytic Ser of thrombin (the enzyme) and by exposing the Arg residue at the site of activation of protein C (the substrate). Studies under specific aim 1 will pursue X-ray, single molecule Förster resonance energy transfer and small angle X-ray spectroscopy of protein C free and bound to FP. Additional details on the conformation of protein C and of its activation domain in solution will be obtained by NMR measurements of miniPC. Success of these studies will provide unprecedented and much needed structural information on protein C and will significantly expand our understanding of the role of conformational plasticity in the mechanism of zymogen activation in this and other members of the trypsin family. Structural studies will be complemented by mutagenesis studies under specific aim 2. The effect of thrombomodulin on the catalytic Ser of thrombin will be investigated either directly through Thr, Cys and Tyr substitutions, or indirectly by removal of potential steric hindrance in the active site region. The effect of thrombomodulin on the site of cleavage of the activation domain of protein C will be investigated with substitutions that disengage the side chain of R169 from neighbor interactions through perturbation of backbone and side chains. Success of our studies will advance our basic knowledge on a key regulatory reaction of the coagulation cascade and will offer a relevant template for the analysis of other cofactor-assisted interactions in the blood coagulation, complement and fibrinolytic cascades.

抽象的 拟议的研究项目重点关注凝血酶对蛋白 C 的血栓调节蛋白依赖性激活 作为凝血反应的关键调节反馈回路，我们对这种反应的兴趣源于它。 生理相关性，缺乏对其机制和转化的分子理解 基础知识的进步可能带来机遇。 我们最近在凝血酶原结晶及其溶液结构表征方面取得了成功 如实验室开发的新试剂，即不含辅助 Gla 和 EGF 的蛋白 C 衍生物 在大肠杆菌中表达的用于同位素标记的结构域（miniPC）和凝血酶的融合蛋白（FP） 血栓调节蛋白的 EGF456 结构域概括了凝血酶的结构和功能特性 我们的假设指导是血栓调节蛋白（辅助因子）通过优化发挥作用。 凝血酶（酶）的催化 Ser 的环境，并通过暴露在该位点的 Arg 残基 特定目标 1 下的研究将追求 X 射线、单分子 Förster。 游离和与 FP 结合的蛋白质 C 的共振能量转移和小角 X 射线光谱。 溶液中蛋白质 C 的构象及其激活结构域的详细信息将通过 NMR 获得 这些研究的成功将提供前所未有的、急需的结构。 有关蛋白质 C 的信息，并将显着扩展我们对构象可塑性作用的理解 该酶和胰蛋白酶家族其他成员的酶原激活机制将得到结构研究。 2. 血栓调节蛋白对催化作用的影响 凝血酶的 Ser 将直接通过 Thr、Cys 和替代 Tyrs 进行研究，或通过间接研究 消除活性位点区域中的潜在空间位阻。 将通过脱离侧面的取代来研究蛋白 C 激活结构域的切割 R169 链通过主链和侧链的扰动进行邻居相互作用。 研究将增进我们对凝血级联关键调节反应的基础知识，并将提供 用于分析凝血过程中其他辅因子辅助相互作用的相关模板， 补体和纤溶级联。