Mechanism of Staphylocoagulase-activated Blood Clotting

描述（由申请方提供）：拟定研究的广泛目标是确定S.金黄色葡萄球菌蛋白、葡萄球菌凝固酶（SC），以及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，并诱导酶原中功能性催化位点的形成，而无需通常严格要求的肽键裂解。假设这种独特的构象激活机制涉及SC和Pro的初始相遇，随后在两个或更多个离散的构象变化中激活催化位点和占据调节性proexosite I。该机制可能涉及proexosite I占领和催化位点激活，稳定SC的活性构象的高亲和力结合之间的构象连接，是不太可能需要插入的SC氨基末端到一个结合口袋中的Pro催化结构域。SC-Pro将纤维蛋白原（Fbg）转化为纤维蛋白（Fbn）的独特特异性的潜在机制被假设为绕过并抑制Pro活化的正常反应。这种机制是心内膜炎心脏瓣膜上血小板-Fbn-细菌赘生物繁殖的核心。假设SC-Pro/T复合物的Fbg clofting活性涉及通过SC-Pro/T复合物上Fbg结合外位点的表达特异性识别Fbg作为底物，以及催化位点特异性的变化。生物化学，生物物理，和结构的方法，采用新的活性位点标记的荧光衍生物的Pro提出了测试假设的构象激活的Pro SC和其特异性的基础上转换成Fbg FBN。具体目标是：（1）确定SC激活Pro构象的热力学机制;（2）确定构象激活过程中各个分子事件的动力学途径;（3）阐明SC-Pro/T复合物特异性识别Fbg的机制;（4）确定SCI-327与Pro/T结合的三维结构。这些研究对于理解SC如何规避丝氨酸蛋白酶酶原激活中对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，并可能最终允许基于抑制SC激活的血液凝固来开发抗生素的治疗。

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.