Mechanism of Staphylocoagulase-activated Blood Clotting

描述（由申请人提供）：拟议研究的总体目标是确定金黄色葡萄球菌蛋白、葡萄球菌凝固酶（SC）激活人血液凝固的分子机制，以及该机制在心内膜炎发病机制中的作用。SC与人凝血酶原（Pro）紧密结合，诱导酶原中功能催化位点的形成，而不需要通常严格要求的肽键断裂。这种独特的构象激活机制被假设为涉及SC和Pro的初始相遇，随后在两个或多个离散构象变化中激活催化位点和占据调节原外生体I。其机制可能涉及原外生体I占据和催化位点激活之间的构象连接，通过SC与活性构象的高亲和力结合来稳定，并且不太可能需要将SC氨基末端插入到Pro催化结构域的结合口袋中。SC-Pro将纤维蛋白原（Fbg）转化为纤维蛋白（Fbn）的独特特异性的机制被假设为绕过和抑制Pro激活的正常反应。这一机制是心内膜炎患者心脏瓣膜上血小板- fbn细菌繁殖的核心机制。据推测，SC-Pro/T复合物的Fbg凝结活性除了催化位点特异性的变化外，还涉及通过在SC-Pro/T复合物上表达Fbg结合外源位点对Fbg作为底物的特异性识别。提出了采用新的活性位点标记的Pro荧光衍生物的生化、生物物理和结构方法来验证SC对Pro构象激活的假设以及其将Fbg转化为Fbn的特异性的基础。具体目的是：(1)确定SC活化Pro构象的热力学机理；(2)确定构象活化中单个分子事件的动力学途径；(3)阐明SC-Pro/T配合物特异性识别Fbg的机制；(4)确定与Pro/T物种结合的SCI-327的三维结构。所提出的研究对于理解SC如何绕过丝氨酸蛋白酶酶原激活中肽键切割的严格要求具有重要意义。这些研究将为SC激活Pro在心内膜炎发病机制中的作用提供新的见解，并可能最终允许开发基于抑制SC激活的凝血的抗生素辅助治疗。

DESCRIPTION (provided by applicant): The broad goal of the proposed studies is to define the molecular mechanism of the activation of human blood coagulation by the S. aureus protein, staphylocoagulase (SC), and the role of the mechanism in the pathogenesis of endocarditis. SC binds tightly to human prothrombin (Pro) and induces formation of a functional catalytic site in the zymogen without the usual strictly required peptide bond cleavages. This unique conformational activation mechanism is hypothesized to involve initial encounter of SC and Pro, followed by activation of the catalytic site and occupation of regulatory proexosite I in two or more discrete conformational changes. The mechanism may involve conformational linkage between proexosite I occupation and catalytic site activation, stabilization by high affinity binding of SC to the active conformation, and is unlikely to require insertion of the SC amino-terminus into a binding pocket in the Pro catalytic domain. The mechanism underlying the unique specificity of SC-Pro to convert fibrinogen (Fbg) to fibrin (Fbn) is hypothesized to bypass and inhibit the normal reactions of Pro activation. This mechanism is central to the propagation of platelet- Fbn-bacteria vegetations on heart valves in endocarditis. Fbg clofting activity of the SC-Pro/T complexes is hypothesized to involve specific recognition of Fbg as a substrate through expression of a Fbg-binding exosite on the SC-Pro/T complexes, in addition to changes in catalytic site specificity. Biochemical, biophysical, and structural approaches employing novel active site-labeled fluorescent derivatives of Pro are proposed to test hypotheses for conformational activation of Pro by SC and the basis of its specificity for conversion of Fbg to Fbn. Specific Aims are: (1) To determine the thermodynamic mechanism of conformational activation of Pro by SC; (2) To define the kinetic pathway of individual molecular events in conformational activation; (3) To elucidate the mechanism of specific recognition of Fbg as a substrate of SC-Pro/T complexes; and (4) To determine the three dimensional structures of SCI-327 bound to Pro/T species. The proposed studies are of fundamental significance in understanding how SC can circumvent the otherwise strict requirement for peptide bond cleavage in serine proteinase zymogen activation. The studies will provide new insight into the role of activation of Pro by SC in the pathogenesis of endocarditis and may ultimately allow therapy adjunctive to antibiotics to be developed based on inhibition of SC-activated blood coagulation.