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

描述（由申请人提供）：双组分信号（TCS）系统是环境变化生物传感的主要形式，在真细菌和其他生物体中发现了超过45，000个TCS系统。TCS途径调节广泛的病原性细菌和真菌的感染性、毒力和耐药性，但在哺乳动物中没有发现，这使它们成为抗菌剂开发的主要目标。顾名思义，TCS系统至少由两种类型的蛋白质组成：组氨酸激酶（HK），其将环境刺激的变化转化为催化活性水平的改变，以及反应调节剂 (RRs)其被HK磷酸化并随后被激活以执行生物反应。虽然几个TCS系统已作为信号转导研究的范例，但两个基本步骤的机制仍不清楚：1）。信号检测是如何通过HK中的感觉和催化域传递的; 2）。磷酸化诱导的结构和动态变化如何在RR接收域和输出域之间传播。在这里，我们解决这些差距与一个综合和全面的研究计划，以检查这样的变构控制TCS系统从保守的应激反应途径。基于生理上的重要性和实验的易处理性，我们集中在光调节TCS蛋白从Erythrobacter litoralis，集中在两个HK（EL 346，EL 368）和他们的RR底物（R1，R5）。利用光作为一种已知的和容易操纵的刺激，再加上这些HK和RR蛋白质对结构和功能研究的出色适用性，我们有一个独特的机会来研究三个目标：通过检查光敏传感器结构域如何控制组氨酸激酶活性来测试HK调节模型; 2）.确定激活对激酶下游的反应调节剂的影响; 3）.确定这些HK和RR蛋白中结构引导突变的体内效应。我们将使用生物物理方法的组合-包括溶液NMR光谱和X射线晶体学-来表征这些蛋白质和复合物的结构，动力学和相互作用。这些结构模型的见解将与体外和基于细胞的功能测定的组合进行测试。这项研究的结果将包括有关这些蛋白质所采用的基本调控过程的信息，这些信息将广泛适用于信号转导研究。

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.