Assembly and Function of Bacterial Chemotaxis Receptor Signaling Complexes

描述（由申请人提供）：大肠杆菌的趋化信号转导途径是分布在细菌和古细菌中的一大家族信号转导途径的代表。众所周知，这些途径介导自由游动和表面相关细菌的趋化性和光性，但越来越多的人认识到，它们调节多细胞聚集、菌毛表达和生物膜形成——这些医学上相关的现象是病原微生物的特征。大肠杆菌系统的相对简单性，加上其研究的发达和复杂的工具，为详细确定大肠杆菌途径的感觉生理学、生物化学和结构生物学提供了强有力的理论依据。大肠杆菌系统中的跨膜信号发生在与细胞质接头蛋白（CheW）和中央信号激酶（CheA）相关的受体蛋白的异质簇或阵列的背景下。引诱剂与受体结合会引起受体的细微构象变化，这种变化以某种方式在膜上传播，产生两种作用(i)抑制CheA， （ii）刺激受体甲基化。激酶抑制表现出积极的协同性，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在更复杂的样品（体外到体内）中与信号蛋白的相互作用，以确定哪些结构和相互作用对激酶和甲基化活性的控制至关重要。最简单的系统，胞质结构域的活性复合物，CheA和CheW组装在囊泡表面，将进行最详细的研究，结合生化和生物物理工具（活性测定，二硫交联，荧光和固态核磁共振）。对囊泡中完整受体复合物组装的研究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，体内荧光研究将研究功能过程中受体复合物和阵列的重塑。所开发的方法和获得的见解将适用于其他系统，其中构象和亚基关联的变化在跨膜信号传导机制中发挥作用。

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.