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: 10580973
• 负责人: Lynmarie K. Thompson
• 金额: $ 8.03万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-19 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10580973
• 关键词: Antibiotics; Bacteria; Binding; Biochemical; Biological Models; Biology; Catalysis; Cells; Chemoreceptors; Chemotaxis; Complex; Coupling; Cytoplasmic Receptors; Cytoplasmic Tail; Detection; Deuterium; Development; Disease; Environment; Escherichia coli; Exhibits; Goals; Hydrogen; Infection; Isotope Labeling; Left; Ligand Binding; Ligands; Mammals; Maps; Mass Spectrum Analysis; Measurement; Measures; Medicine; Membrane; Methods; Methylation; Molecular; Nanoarray Analytical Device; Pattern; Phosphotransferases; Play; Preparation; Process; Prokaryotic Cells; Protein Fragment; Protein Kinase; Protein Region; Proteins; Receptor Signaling; Rest; Role; Sampling; Signal Transduction; Structural Models; Structure; Swimming; System; Tertiary Protein Structure; Testing; Vesicle; alpha helix; biophysical properties; demethylation; dimer; experimental study; flexibility; insight; interest; novel; pathogen; periplasm; protein complex; receptor; solid state nuclear magnetic resonance

Project Summary/Abstract The overall objective of the project is to determine the mechanism by which bacterial chemotaxis receptors regulate the activity of the central kinase CheA. Bacterial chemotaxis is a well-studied two-component signaling system and is also essential for infection by some pathogens. Two-component signaling systems are widespread in prokaryotes but not found in mammals, making chemotaxis proteins potential targets for novel antibiotics. The specific objective is to determine the molecular details of the interaction between chemoreceptors and CheA, and how these change to control kinase activity. Chemoreceptors function within large membrane-bound hexagonal arrays of receptors, CheA, and CheW. Aims 1-2 will investigate native-like functional arrays of the E. coli Asp receptor cytoplasmic fragment (CF), CheA, and CheW assembled on vesicles. Solid-state NMR methods for selective detection of protein interfaces and rigid protein regions will determine structure and structural changes at the receptor/CheA interface. Complementary hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments will determine how CheA domain interactions and linker flexibility change with signaling state. Aim 3 will first optimize preparations of functional arrays of intact receptors with CheA and CheW, and then apply HDX-MS and selected NMR experiments to this more complex sample to discriminate which signaling-related changes are caused by ligand binding vs receptor methylation (adaptation). These experiments will test our hypothesis that the receptor cytoplasmic domain is partially disordered, and that signaling inputs modulate this disorder to control contacts with CheA and kinase activity. Understanding the mechanism of this key signaling system will yield insights into the roles and mechanisms of disordered domains in other protein complexes and how these contribute to long-range allosteric processes. The project will also demonstrate the promise of combining approaches, such as HDX-MS and solid-state NMR, for advancing mechanistic understanding of the many protein assemblies that play key roles in biology.

项目摘要/摘要 该项目的总体目标是确定细菌趋化受体的机制。 调节中枢肌动蛋白CHEA的活性。细菌趋化性是一种被广泛研究的双组分信号。 而且对于某些病原体的感染也是必不可少的。双分量信令系统广泛使用 在原核生物中没有发现，但在哺乳动物中没有发现，使趋化蛋白成为新型抗生素的潜在靶点。 其具体目标是确定化学受体和CHEA相互作用的分子细节， 以及它们是如何改变以控制激酶活性的。化学感受器在大分子膜结合中的作用 六角形的感受器阵列，CHEA，和咀嚼。AIMS 1-2将研究E。 ColiAsp受体胞浆片段(Cf)、CheA和咀嚼组装在囊泡上。固态核磁共振 选择性检测蛋白质界面和刚性蛋白质区域的方法将决定结构和 受体/CHEA界面的结构变化。互补的氢-氚交换质量 光谱分析(HDX-MS)实验将确定CHEA结构域相互作用和连接子灵活性如何变化 具有信令状态。目标3将首先优化完整受体功能阵列的制备，包括CHEA和 咀嚼，然后应用HDX-MS和精选的核磁共振实验来区分这个更复杂的样品 与信号相关的变化是由配体结合与受体甲基化(适应)引起的。这些 实验将验证我们的假设，即受体细胞质区域部分无序，并且 信号输入调节这种紊乱，以控制与CHEA和激酶活性的接触。 了解这一关键信号系统的机制将有助于深入了解 其他蛋白质复合体中的无序结构域以及这些结构域如何促成长程变构过程。这个 该项目还将展示组合方法的前景，如HDX-MS和固态核磁共振，用于 促进对在生物学中起关键作用的许多蛋白质组合的机械理解。