Structure and function of membrane receptor signaling complex in bacterial chemot

描述(申请人提供)：细菌细胞通过将化学配体与膜受体结合，激活多蛋白受体信号复合体，并产生可扩散的细胞内信号，最终调节鞭毛马达的旋转，对其化学环境的变化(趋化性)做出反应。大肠杆菌中的趋化信号通路已成为生物学中最具特征的信号转导网络。所有负责激发和适应的蛋白质组分都已经被鉴定和表征，并且它们的可溶性域结构被确定为原子分辨率。也有大量关于趋化信号的突变、化学交联和GFP标记研究。然而，由受体、激酶CHEA和偶联蛋白Chew组成的基本信号单位的结构仍然不清楚，这是理解信号转导的分子机制所必需的。这项建议中描述的研究旨在获得三元受体信号复合体的高分辨率结构，以及它在完整细胞中的高阶组装结构，主要是通过高分辨率三维冷冻电子显微镜。我们还将使用结构方法和功能分析来表征受体激活/失活时复合体的构象变化。这些结构与功能和生化分析相结合，有望提供关于微小的外部化学信号如何传递到组氨酸激酶并通过天然细胞内的信号复合体的大量组装而放大的详细了解。这些结果也将为建立受体信号系统的计算模型提供基础，因为大肠杆菌趋化性一直是理解信号转导和信号处理的分子机制的理想模型系统。公共卫生相关性：细菌趋化中的刺激-反应耦合机制已成为理解细菌和真核细胞中细胞内信号转导原理的范例。大肠杆菌趋化性也是高度保守的“双组分系统”的代表，它控制着原核生物从细胞分化和发育到昼夜节律和发病机制的各种过程，包括真细菌和古生物。这些系统都含有两种中心酶，一种是组氨酸蛋白激酶，另一种是调节微生物和植物中的磷信号转导网络的反应调节因子。此外，细菌的趋化反应对细菌的定植和感染至关重要，而介导这种反应的信号转导系统是抗菌药物开发的潜在靶点。因此，深入了解细菌趋化作用中的信号机制对生物学和医学的许多领域都具有重要意义。我们提出的对趋化受体信号复合体和复合体阵列进行全面和集成的结构和功能分析的努力将有助于深入了解受体-激酶相互作用、信号复合体的形成、感觉簇的形成和构象耦合，并有助于从总体上更好地理解信号转导和信号处理。

DESCRIPTION (provided by applicant): Bacterial cells respond to the changes in their chemical environment (chemotaxis) by binding of the chemical ligand to membrane receptors, activating the multi-protein receptor signaling complex, and generating a diffusible intracellular signal that ultimately regulates the rotation of the flagella motor. The chemotactic signaling pathway in E. coli has emerged as the best-characterized signal transduction network in biology. All the protein components responsible for excitation and adaptation have been identified and characterized, and their soluble domain structures determined to atomic resolution. There have also been numerous mutagenesis, chemical cross-linking and GFP-tagging studies on chemotaxis signaling. However, the structures of the basic signaling unit consisting of the receptor, the kinase CheA and coupling protein CheW, that are essential to understand the molecular mechanism of the signal transduction remain elusive. The studies described in this proposal are targeted to obtaining high resolution structures of the ternary receptor signaling complex, as well as structures of its higher order assembly in intact cells, primarily by high resolution three-dimensional cryo- electron microscopy. We will also characterize the conformational changes of the complex upon receptor activation/inactivation using both structural methods and functional assays. These structures, combining with functional and biochemical analysis, are expected to provide a detailed understanding on how minute external chemical signals are transmitted to the histidine kinase and amplified through a large assembly of signaling complexes within the native cells. These results will also provide a foundation for generating computational models of receptor signaling systems, since the E. coli chemotaxis has been an ideal model system for understanding the molecular mechanisms of signal transduction and signal processing in general. PUBLIC HEALTH RELEVANCE: The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding the principles of intracellular signal transduction both in bacterial and eukaryotic cells. E. coli chemotaxis is also a representative of the highly conserved "two-component systems" that control processes ranging from cell differentiation and development to circadian rhythms and pathogenesis in prokaryotes including both eubacterial and archaeal species. These systems all contain two central enzymes, a histidine protein kinase and a response regulator that mediates phosphor relay signal transduction networks in microorganisms and plants. In addition, bacterial chemotaxis response is crucial for colonization and infection, and the signal transduction systems that mediate such responses are potential targets for antimicrobial drug development. A deeper understanding of the mechanism of signaling in bacterial chemotaxis is therefore of great interests for many areas of biology and medicine. Our proposed efforts for a comprehensive and integrated structural and functional analysis of chemotaxis receptor signaling complexes and complex arrays will provide insight into receptor-kinase interaction, signaling complex formation, sensory cluster formation and conformation coupling, and contribute to a better understanding of the signal transduction and signal processing in general.