Assembly and Function of Bacterial Chemotaxis Receptor Signaling Complexes

描述(申请人提供)：大肠杆菌的趋化信号转导通路是分布在细菌和古生菌中的一大类信号转导通路的代表。这些途径已经知道在自由游泳和表面结合的细菌中介导趋化性和趋光性，但越来越多的人认识到它们可以调节多细胞聚集、菌毛表达和生物膜的形成--这些现象在医学上是病原微生物的特征。大肠杆菌系统的相对简单，加上其研究的成熟和复杂的工具，为详细确定大肠杆菌途径的感觉生理、生物化学和结构生物学提供了强有力的理论基础。大肠杆菌系统中的跨膜信号发生在与细胞质适配器蛋白CHEW和中央信号转导蛋白CHEA相关的异质受体蛋白簇或阵列的背景下。吸引剂与受体的结合导致受体的微妙构象变化，这些变化以某种方式传播到膜上，产生两种效果：(1)抑制CHEA，(2)刺激受体甲基化。激酶抑制表现出正的协作性，表明受一组受体的调节。理解详细机制的关键在于了解这些受体复合体簇在功能过程中组装和重塑的方式。在这个项目中，我们将研究受体之间的相互作用以及与信号蛋白之间的相互作用，以确定哪些结构和相互作用对控制激酶和甲基化活性至关重要。我们将结合生物化学和生物物理工具(活性分析、二硫键交联、荧光和固体核磁共振)，对最简单的系统，即组装在囊泡表面的细胞质结构域的活性复合体CHEA和CHEW进行最详细的研究。对囊泡中完整受体复合体组装的研究将确定阵列中的相互作用如何受到其他受体结构域和配体的调节。最后，体内荧光研究将研究受体复合体和阵列在功能过程中的重塑。发展的方法和获得的见解将适用于其他系统，在这些系统中，构象和亚单位结合的变化在跨膜信号机制中发挥作用。 公共卫生相关性：该项目试图通过使用重组为功能信号复合体和活细菌细胞的分离蛋白成分来确定信号蛋白在大肠杆菌趋化途径中的功能。这些样品将用生化和生物物理技术相结合的方法进行测试，以将结构与活性联系起来。这个大肠杆菌系统代表了包括致病细菌在内的微生物中的一大类信号通路，因此，通过这个项目过程中获得的信息将与大量的通路相关，这可能有助于新型抗菌剂的开发。

DESCRIPTION (provided by applicant): The chemotactic signal transduction pathway of Escherichia coli is representative of a large family of signal transduction pathways that are distributed throughout the Bacteria and Archaea. These pathways are already known to mediate chemotaxis and phototaxis in free-swimming and surface-associated bacteria, but are being recognized increasingly to regulate multicellular aggregation, pilus expression, and biofilm formation - medically relevant phenomena that are characteristic of pathogenic microbes. The relative simplicity of the E. coli system, combined with the well-developed and sophisticated tools for its study, provides a strong rationale to determine in detail the sensory physiology, biochemistry and structural biology of the E. coli pathway. Transmembrane signaling in the E. coli system occurs in the context of a heterogeneous cluster, or array, of receptor proteins that are associated with the cytoplasmic adaptor protein, CheW, and the central signaling kinase, CheA. Attractant binding to the receptors causes subtle conformational changes in the receptor that are somehow propagated across the membrane to produce two effects (i) inhibition of CheA, and (ii) stimulation of receptor methylation. Kinase inhibition exhibits a positive cooperativity indicative of regulation by a cluster of receptors. The key to understanding the detailed mechanism lies in understanding the manner in which these clusters of receptor complexes are assembled and remodeled during function. In this project we will investigate the interactions among receptors and with the signaling proteins in samples of successively greater complexity (in vitro to in vivo), to determine what structures and interactions are critical to the control of kinase and methylation activity. The simplest system, active complexes of the cytoplasmic domain, CheA, and CheW assembled on vesicle surfaces, will be studied in the greatest detail, with a combination of biochemical and biophysical tools (activity assays, disulfide crosslinking, fluorescence, and solid-state NMR). Studies of assemblies of complexes of the intact receptor in vesicles will determine how the interactions in the array are modulated by the other receptor domains and by ligand. Finally, in vivo fluorescence studies will investigate remodeling of receptor complexes and arrays during function. The approaches developed and insights gained will be applicable to other systems in which changes in both conformation and subunit association play a role in the mechanism of transmembrane signaling. PUBLIC HEALTH RELEVANCE: This project seeks to determine the function of signaling proteins in the chemotaxis pathway of E. coli through the use of isolated protein components reassembled into functioning signaling complexes and living bacterial cells. These samples will be tested with a combination of biochemical and biophysical techniques to correlate structure with activity. This E. coli system is representative of a large family of signaling pathways in microbes, including disease-causing bacteria, and therefore, the information obtained through the course of this project will be relevant to a great number of pathways, which may benefit the development of novel antimicrobial agents.