Assembly and Function of Bacterial Chemotaxis Receptor Signaling Complexes

细菌趋化性受体信号复合物的组装和功能

• 批准号: 8054260
• 负责人: Lynmarie K. Thompson
• 金额: $ 31.28万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054260
• 关键词: Adaptor Signaling Protein; Address; Affinity; Archaea; Bacteria; Binding; Biochemical; Biochemistry; Biological Assay; Biology; Cells; Characteristics; Chemicals; Chemoreceptors; Chemotaxis; Complex; Cytoplasmic Tail; Development; Dimerization; Disease; Disulfides; Equilibrium; Escherichia coli; Exhibits; Family; Family member; Fluorescence; Fluorescence Resonance Energy Transfer; In Vitro; Label; Lateral; Life; Ligand Binding; Ligand Binding Domain; Ligands; Lipid Bilayers; Lipids; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Methodology; Methylation; Microbe; Microbial Biofilms; Modification; Molecular; Molecular Conformation; Monitor; Pathway interactions; Phosphotransferases; Physiological; Pilum; Play; Process; Property; Protein Dynamics; Proteins; Protons; Receptor Signaling; Regulation; Relative (related person); Research; Role; Rotation; Sampling; Sensory; Sensory Physiology; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Stimulus; Structure; Surface; Swimming; System; Techniques; Testing; Validation; Vesicle; antimicrobial drug; crosslink; density; dimer; in vivo; insight; mutant; novel; overexpression; periplasm; public health relevance; receptor; reconstitution; research study; response; solid state nuclear magnetic resonance; stoichiometry; structural biology; tool

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.

描述（由申请方提供）：大肠埃希菌的趋化性信号转导途径是分布于细菌和大肠杆菌中的信号转导途径大家族的代表。已知这些途径介导自由游动和表面相关细菌中的趋化性和趋光性，但越来越多地被认识到调节多细胞聚集、菌毛表达和生物膜形成-病原微生物特有的医学相关现象。E.大肠杆菌系统的建立，结合成熟的研究工具，为详细研究大肠杆菌的感觉生理、生物化学和结构生物学提供了强有力的理论基础。coli途径。E.大肠杆菌系统发生在与细胞质衔接蛋白CheW和中枢信号激酶CheA相关的受体蛋白的异质簇或阵列的背景下。引诱剂与受体的结合引起受体的细微构象变化，这些变化以某种方式跨膜传播以产生两种效应：（i）抑制CheA，和（ii）刺激受体甲基化。激酶抑制表现出正协同性，表明受一组受体的调节。了解详细机制的关键在于了解这些受体复合物簇在功能期间组装和重塑的方式。在这个项目中，我们将研究受体之间的相互作用，并与信号蛋白在样品中的连续更大的复杂性（在体外到体内），以确定什么结构和相互作用是关键的激酶和甲基化活性的控制。最简单的系统，活性复合物的细胞质结构域，CheA，和CheW组装在囊泡表面，将进行最详细的研究，结合生物化学和生物物理工具（活性测定，二硫键交联，荧光和固态NMR）。研究囊泡中完整受体复合物的组装将确定阵列中的相互作用如何被其他受体结构域和配体调节。最后，在体内荧光研究将调查功能过程中受体复合物和阵列的重塑。开发的方法和获得的见解将适用于其他系统中，在构象和亚基协会的变化发挥作用的跨膜信号的机制。 公共卫生相关性：该项目旨在确定大肠杆菌趋化途径中信号蛋白的功能。大肠杆菌通过使用分离的蛋白质成分重新组装成功能信号复合物和活的细菌细胞。这些样品将结合生物化学和生物物理技术进行测试，以将结构与活性相关联。这个E。大肠杆菌系统是微生物包括致病细菌中信号通路的一大家族的代表，因此，通过本项目的过程中获得的信息将与大量的通路相关，这可能有利于开发新的抗菌剂。