Molecular mechanism of protein C activation

摘要 拟议的研究项目集中在凝血酶对蛋白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的 生理相关性，缺乏对其机制的分子理解和翻译 基础知识的进步可能带来的机会。蛋白质C的研究动机是 我们最近在凝血酶原的结晶及其在溶液中的结构表征方面的成功，以及 如通过实验室中开发的新试剂，即，缺乏辅助性Gla和EGF的蛋白C衍生物 结构域（miniPC）在E.大肠杆菌进行同位素标记，以及凝血酶与 血栓调节蛋白的EGF 456结构域，其概括了凝血酶的结构和功能特性， 血栓调节蛋白复合物我们的指导假设是血栓调节蛋白（辅因子）通过优化 凝血酶（酶）的催化Ser的环境，并通过暴露Arg残基的位点， 蛋白C（底物）的活化。具体目标1下的研究将追求X射线、单分子Förster 蛋白C游离和与FP结合的共振能量转移和小角X射线光谱。额外 蛋白C的构象及其在溶液中的活化结构域的细节将通过NMR获得 miniPC的测量。这些研究的成功将提供前所未有的和急需的结构性 蛋白质C的信息，并将显着扩大我们的构象可塑性的作用的理解 在胰蛋白酶家族的这个和其他成员中的酶原激活机制中。结构研究将 通过具体目标2下的诱变研究加以补充。血栓调节蛋白对血管内皮细胞催化 凝血酶的Ser将通过Thr、Cys和Tyr取代直接研究，或通过 去除活性部位区域中潜在的空间位阻。血栓调节蛋白对血栓形成部位的影响 蛋白C的激活结构域的切割将被研究， 通过骨架和侧链的扰动，R169链与邻近相互作用。成功的 研究将推进我们对凝血级联反应的关键调节反应的基础知识，并将提供 用于分析血液凝固中其他辅因子辅助相互作用的相关模板， 补体和纤维蛋白溶解级联。

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.