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的依赖血栓调节蛋白的激活 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，缺乏对其机制和翻译的分子理解 基础知识的进步可能带来的机会。对蛋白C的研究受到以下因素的推动 我们最近成功地结晶了凝血酶原并表征了其溶液结构 通过实验室开发的新试剂，即不含辅助GLA和EGF的蛋白C的衍生物 在大肠杆菌中表达的用于同位素标记的结构域(Mini PC)和凝血酶与 凝血酶调节蛋白的EGF456结构域，它概括了凝血酶的结构和功能特性 血栓调节蛋白复合体。我们的指导性假设是血栓调节蛋白(辅因子)通过优化 凝血酶催化序列(酶)的环境和暴露Arg残基在 蛋白C(底物)的激活。在特定目标1下的研究将追求X射线、单分子Förster 蛋白质C游离和与FP结合的共振能量转移和小角X射线光谱。其他内容 将通过核磁共振获得蛋白质C的构象及其在溶液中的激活结构域的详细信息 微型PC的测量。这些研究的成功将提供史无前例和亟需的结构 关于蛋白质C的信息，并将大大扩展我们对构象可塑性作用的理解 在这个和胰酶家族的其他成员的酶原激活机制中。结构研究将 辅以特定目的的突变研究2.血栓调节蛋白对酶促反应的影响 凝血酶的序列将直接通过苏氨酸、半胱氨酸和酪氨酸取代，或间接通过 消除了活性中心区域中的潜在空间位阻。血栓调节蛋白对心肌梗死部位的影响 蛋白C激活域的切割将通过与侧边分离的取代来研究 R169的链通过主干和侧链的扰动来自邻居的相互作用。我们的成功 研究将增进我们对凝固级联的关键调节反应的基本知识，并将提供 用于分析凝血中其他辅因子辅助的相互作用的相关模板， 补体和纤溶级联反应。

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.