Mechanistic principles of signal detection and transmission in bacterial two-comp

描述(申请人提供)：双组分信号(TCS)系统是生物感知环境变化的主要形式，在真细菌和其他生物体中发现了超过45,000个TCS系统。TCS通路调节着广泛的致病细菌和真菌的感染性、毒性和耐药性--但在哺乳动物中找不到，这使它们成为抗菌药物开发的主要靶点。顾名思义，TCS系统最小限度地由两种蛋白质组成：组氨酸激酶(HKs)，它将环境刺激的变化转化为不同水平的催化活性，以及反应调节因子 (RR)被HKS磷酸化，然后被激活以执行生物反应。虽然几个TCS系统已经成为信号转导研究的范例，但两个基本步骤的机制仍然不清楚：1)。在HKS和2)中信号检测是如何通过感觉域和催化域传递的。磷酸化诱导的结构和动态变化是如何在RR接收域和输出域之间传播的。在这里，我们通过一个完整而全面的研究计划来解决这些差距，以从保守的应激反应途径来检查TCS系统中的这种变构控制。基于生理重要性和实验可操作性，我们重点研究了小红杆菌的光调控TCS蛋白，主要集中在两个HKS(EL346，EL368)及其RR底物(R1，R5)上。利用光作为一种已知且易于操作的刺激，再加上这些HK和RR蛋白在结构和功能研究中的出色适用性，我们有一个独特的机会来研究三个目标：1)。通过检测光敏感受器结构域如何控制组氨酸激酶活性来测试HK调控模型；2)确定激活对紧接在激酶下游的反应调节器的影响；3)。确定这些HK和RR蛋白结构导向突变的体内效应。我们将结合使用生物物理方法--包括溶液核磁共振光谱和X射线结晶学--来表征这些蛋白质和复合体的结构、动力学和相互作用。来自这些结构模型的见解将通过体外和基于细胞的功能分析的组合进行测试。这项研究的结果将包括关于这些蛋白质所采用的基本调控过程的信息，提供将广泛适用于信号转导研究的见解。

DESCRIPTION (provided by applicant): Two-component signaling (TCS) systems are a predominant form of biological sensing of environmental change, with over 45,000 TCS systems found in eubacteria and other organisms. TCS pathways regulate infectivity, virulence and resistance in a wide range of pathogenic bacteria and fungi - but are not found in mammals, making them prime targets for antimicrobial development. As suggested by the name, TCS systems are minimally composed of two types of proteins: histidine kinases (HKs) which convert changes in environmental stimuli into altered levels of catalytic activity, and response regulators (RRs) that are phosphorylated by HKs and subsequently activated to execute biological responses. While several TCS systems have served as paradigms of signal transduction studies, the mechanisms of two fundamental steps are still unclear: 1). how signal detection is relayed through sensory and catalytic domains in HKs and 2). how phosphorylation-induced structural and dynamic changes are propagated between RR receiver and output domains. Here we address these gaps with an integrated and comprehensive research plan to examine such allosteric control in TCS systems from a conserved stress response pathway. Based on physiological importance and experimental tractability, we have focused on light-regulated TCS proteins from Erythrobacter litoralis, concentrating on two HKs (EL346, EL368) and their RR substrates (R1, R5). Taking advantage of light as a known and easily manipulated stimulus, coupled with the excellent suitability of these HK and RR proteins for structural and functional studies, we have a unique opportunity to examine three goals: 1). Testing a model of HK regulation by examining how photosensitive sensor domains control histidine kinase activity; 2). Establishing the effects of activation on response regulators immediately downstream from the kinases; 3). Determining the in vivo effects of structure-guided mutations in these HK and RR proteins. We will use a combination of biophysical methods - including solution NMR spectroscopy and X-ray crystallography - to characterize the structure, dynamics and interactions of these proteins and complexes. Insights from these structural models will be tested with a mix of in vitro and cell-based functional assays. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights that will be broadly applicable for signal transduction studies.