Assembly and Function of Bacterial Chemotaxis Receptor Signaling Complexes

项目总结/摘要 该项目的总体目标是确定细菌趋化性受体 调节中枢激酶CheA的活性。细菌的趋化性是一种被广泛研究的双组分信号 它也是某些病原体感染所必需的。双组分信号系统广泛应用 在原核生物中发现，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。 具体目标是确定化学感受器和CheA之间相互作用的分子细节， 以及它们如何改变以控制激酶活性。化学感受器在大的膜结合的 六边形排列的受体，CheA和CheW。目的1-2将研究E. coli Asp受体胞质片段（CF）、CheA和CheW组装在囊泡上。固态NMR 用于选择性检测蛋白质界面和刚性蛋白质区域的方法将确定结构， 受体/CheA界面的结构变化。互补氢氘交换质量 光谱分析（HDX-MS）实验将确定CheA结构域相互作用和接头柔性如何变化 信号状态。目的3将首先优化完整受体与CheA的功能阵列的制备， CheW，然后将HDX-MS和选定的NMR实验应用于该更复杂的样品以区分 其信号相关的变化是由配体结合相对于受体甲基化（适应）引起的。这些 实验将检验我们的假设，即受体胞质结构域部分无序， 信号输入调节这种疾病以控制与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 其他蛋白质复合物中的无序结构域以及这些结构域如何促进长程变构过程。的 该项目还将展示结合方法的前景，如HDX-MS和固态NMR， 推进对许多在生物学中起关键作用的蛋白质组装的机械理解。

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.