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: 10655555
• 负责人: Lynmarie K. Thompson
• 金额: $ 35.21万
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655555
• 关键词: Antibiotics; Bacteria; Binding; Biochemical; Biological Models; Biology; Catalysis; Cells; Chemoreceptors; Chemotaxis; Complex; Coupling; Cytoplasm; 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 Array; Protein Fragment; 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; receptor function; 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 该项目的总体目的是确定细菌趋化受体的机制 调节中央激酶CHEA的活性。细菌趋化性是一种经过良好研究的两种组成信号传导 系统，对于某些病原体感染也是必不可少的。两个组件信号传导系统广泛 在原核生物中，但在哺乳动物中未发现，使趋化蛋白具有新型抗生素的潜在靶标。 具体目标是确定化学感受器与CHEA之间相互作用的分子细节， 以及这些如何改变以控制激酶活性。化学感受器在大膜结合内发挥作用 六角形的受体，CHEA和咀嚼。 AIMS 1-2将研究E的类似天然的功能阵列。 大肠子ASP受体细胞质片段（CF），CHEA和咀嚼组装在囊泡上。固态NMR 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界面处的结构变化。补充氢氘交换质量 光谱法（HDX-MS）实验将决定CHEA域相互作用和链接器灵活性如何变化 具有信号状态。 AIM 3将首先优化具有CHEA和CHEA完整受体功能阵列的制备 咀嚼，然后将HDX-MS和选定的NMR实验应用于此更复杂的样品以区分 哪些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 实验将检验我们的假设，即受体细胞质结构域是部分混乱的，并且 信号传导输入调节这种疾病以控制与CHEA和激酶活性的接触。 了解此关键信号系统的机制将产生对角色和机制的见解 其他蛋白质复合物中的无序结构域以及它们如何促进远程变构过程。这 项目还将证明将方法（例如HDX-MS和固态NMR）组合的希望 促进对许多在生物学中起关键作用的蛋白质组件的理解。

项目成果

Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA.

• DOI: 10.1073/pnas.2218467120
• 发表时间: 2023-08-08
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Tran, Thomas;Mudiyanselage, Aruni P. K. K. Karunanayake;Eyles, Stephen J.;Thompson, Lynmarie K.
• 通讯作者: Thompson, Lynmarie K.

Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.

基于焦碳酸二乙酯的膜复杂系统中蛋白质相互作用的共价标记质谱分析。

• DOI: 10.1021/jasms.2c00262
• 发表时间: 2023
• 期刊: Journal of the American Society for Mass Spectrometry
• 影响因子: 3.2
• 作者: Pan,Xiao;Tran,Thomas;Kirsch,ZacharyJ;Thompson,LynmarieK;Vachet,RichardW
• 通讯作者: Vachet,RichardW

Protein rings are critical to the remarkable signaling properties of bacterial chemotaxis nanoarrays.

• DOI: 10.1126/scisignal.abn2056
• 发表时间: 2022-01-25
• 期刊: Science signaling
• 影响因子: 7.3
• 作者: Thompson LK

Carbon-nitrogen REDOR to identify ms-timescale mobility in proteins.

碳-氮 REDOR 用于识别蛋白质中毫秒时间尺度的迁移率。

• DOI: 10.1016/j.jmr.2019.05.008
• 发表时间: 2019
• 期刊: Journal of magnetic resonance (San Diego, Calif. : 1997)
• 作者: Kashefi,Maryam;Malik,Nikita;Struppe,JochemO;Thompson,LynmarieK
• 通讯作者: Thompson,LynmarieK