Mechanism of Staphylocoagulase-activated Blood Clotting

项目摘要。拟议研究的总体目标是通过以下方式确定分子机制： 其中金黄色葡萄球菌蛋白，葡萄球菌凝固酶（SC），激活血液凝固，及其作用 急性细菌性心内膜炎的病理学热力学、动力学和结构方法是 建议解决在理解SC激活的独特机制方面的主要差距 凝血酶原（ProT）非蛋白水解并形成特异性转化纤维蛋白原的SOProT复合物 (Fbg)变成纤维蛋白研究主要集中在以下六个方面：（1）关键供应链的结构-功能关系 将研究诱导构象ProT活化的N-末端插入，以表征 的N-末端结合裂缝的特异性，并评估插入是可逆的假设， 平衡过程，可以诱导不同水平的激活。(2)个体结合的途径， 在SC构象激活ProT的分子机制中的构象变化事件将是 在荧光和活性监测的快速反应动力学研究中定义。(3)结构与功能 Fbg相互作用的两种模式之间的关系，作为活性SC？ProT复合物的底物 通过N-末端SC结构域结合，并通过C-末端SC重复序列作为Fbg粘附蛋白 将在结合、动力学和晶体学研究中进行研究。(4)基于突变的结构-功能 研究将检验人和牛ProT的自溶环残基控制物种的假设 SC-ProT激活的特异性。(5)新发现的蛋白质的机制 SC同系物酶原激活剂和粘附蛋白（ZAAP）家族激活酶原，切割蛋白质 底物，并结合到血浆和细胞外基质蛋白将进行研究。(6)的机理 将研究N-末端序列特异性抗体对SC-ProT活化的抑制作用， 治疗心内膜炎的潜在治疗方法。拟议的研究将提供详细的 了解SC激活血液凝固的分子机制。这可以使设计 治疗心内膜炎的新机制治疗剂。将研究扩展到其他领域 ZAAP家族成员将为这些靶向人类的新型蛋白质的作用提供新的见解。 凝血系统在危及生命的葡萄球菌和链球菌疾病的发病机制。

Project Summary. The overall goal of the proposed studies is to define the molecular mechanism by which the Staphylococcus aureus protein, staphylocoagulase (SC), activates blood coagulation, and its role in the pathology of acute bacterial endocarditis. Thermodynamic, kinetic, and structural approaches are proposed to address major gaps in the understanding of the unique mechanism by which SC activates prothrombin (ProT) non-proteolytically and forms an SOProT complex that specifically converts fibrinogen (Fbg) into fibrin. The studies focus on 6 areas: (1) Structure-function relationships governing the critical SC N-terminal insertion that induces conformational ProT activation will be investigated to characterize the specificity of the N-terminal binding cleft, and to evaluate the hypothesis that insertion is a reversible equilibrium process that can induce different levels of activation. (2) The pathway of individual binding and conformational change events in the molecular mechanism of conformational activation of ProT by SC will be defined in fluorescence- and activity-monitored rapid-reaction kinetic studies. (3) The structure-function relationships between the two modes of Fbg interactions, as a substrate of the active SC¿ProT complex bound via N-terminal SC domains, and as a Fbg adhesion protein through C-terminal SC repeat sequences will be investigated in binding, kinetic, and crystallographic studies. (4) Mutagenesis-based structure-function studies will test the hypothesis that autolysis loop residues of human and bovine ProT control the species specificity of SC-ProT activation. (5) The mechanisms through which proteins of the newly discovered zymogen activator and adhesion p/otein (ZAAP) family of SC homologs activate zymogens, cleave protein substrates, and bind to plasma and extracellular matrix proteins will be investigated. (6) The mechanism of inhibition of SC-ProT activation by antibodies specific for the N-terminal sequence will be investigated as a potential therapeutic approach for treating endocarditis. The studies proposed will provide a detailed understanding of the molecular mechanisms of SC-activated blood coagulation. This may enable the design of new mechanism-based therapeutic agents for treating endocarditis. Extension of the studies to other ZAAP family members will provide new insight into the roles of these novel proteins that target the human blood coagulation system in the pathogenesis of life-threatening staphylococcal and streptococcal diseases.