Sensory Transduction in Bacteria

描述（由申请人提供）：趋氧性是大肠杆菌对氧气的行为反应，由Aer和Tsr受体介导。Aer是一种内部受体，锚定在细胞质膜内部的中间。N-末端PAS结构域具有FAD辅因子，并感测细胞质中的氧化还原和能量水平。Aer的羧基半，也是细胞质的，具有HAMP结构域和在趋化性受体中高度保守的信号传导结构域。阐明Aer信号传导机制将提供PAS和HAMP结构域之间的信号转导的第一个深入研究：重要的感觉模块，广泛存在于生命系统中，但没有被其他研究者广泛研究。具体目标1将研究Aer同源二聚体结构域的结构关系。基于一个结构模型，HAMP结构域被认为是一个四螺旋束，直接与PAS结构域接口，形成Aer的输入/输出模块。HAMP结构域的结构将通过体内二硫键交联和与F. W.达尔奎斯特PAS和HAMP结构域之间的接触表面将使用遗传分析来确定，包括等位基因特异性抑制、表面可及性测量和间二硫键作图。近端信号结构域被认为是一个功能重要的子结构，它将HAMP结构域的旋转信号转化为信号结构域的位移。阐明Aer结构域的结构关系将指导实验设计以确定信号通路。具体目标2将研究Aer同源二聚体内的趋氧性信号传导机制。Aer PAS结构域中FAD的减少被提议引起构象变化，其诱导四螺旋HAMP束的旋转，将Aer信号输出从激酶关闭状态转换为激酶开启状态。将通过计算机模拟分析、位点特异性和随机诱变、FAD的异咯嗪氧化还原中心附近残基的共价标记（使用光标记FAD）和FAD结合测量来鉴定FAD结合裂缝中的关键残基。为了阐明信号通路的序列，将扩展成功的基因内互补研究，以确定锁定在“开启”状态的突变PAS结构域是否通过近端或同源HAMP-AS-2螺旋或两者发出信号。我们还将使用二硫键交联来确定HAMP结构域是否在开和关信号状态下保持稳定的四螺旋束。总之，这些研究应该揭示信号通路的关键残基。特定目标3将确定Aer PAS域感知的信号。虽然需氧性需要电子传递系统，但黄素还原酶（Fre）在体外可降低Aer-FAD。一个重组的趋氧性系统将被用来调查的作用，Free和测试的假设，Aer可以感知细胞质的氧化还原电位和NADH/NAD的比例，独立的电子传递系统。阐明Aer中的信号传导机制应该提供对医学上重要的PAS和HAMP蛋白的感觉机制的深入了解。研究了E.大肠杆菌细菌鉴定出PAS结构域作为受体中的感觉模块。PAS结构域存在于从细菌到人类的1万种蛋白质中，包括医学上重要的蛋白质。这项研究旨在了解感觉受体的功能。所获得的知识将帮助科学家了解更复杂的感觉系统如何监测细胞中的氧浓度和能量水平，如人类神经细胞，如果氧浓度不保持在足够的水平，这些细胞很容易受损。

DESCRIPTION (provided by applicant): Aerotaxis, the behavioral response of Escherichia coli to oxygen, is mediated by the Aer and Tsr receptors. Aer is an internal receptor, anchored in the middle to the inside of the cytoplasmic membrane. An N-terminal PAS domain has a FAD cofactor and senses redox and energy level in the cytoplasm. The carboxyl half of Aer, also cytoplasmic, has a HAMP domain and a signaling domain that is highly conserved in chemotaxis receptors. Elucidating the Aer signaling mechanism will provide the first in-depth study of signal transduction between PAS and HAMP domains: important sensory modules that are widespread in living systems but not widely studied by other investigators. Specific Aim 1 will investigate the structural relationship of the domains of the Aer homodimer. Based on a structural model, the HAMP domain is proposed to be a four-helix bundle that interfaces directly with the PAS domain to form the input/output module of Aer. The structure of the HAMP domain will be determined by disulfide crosslinking in vivo and a NMR solution structure in collaboration with F. W. Dahlquist. Contact surfaces between the PAS and HAMP domains will be determined using genetic analysis, including allele-specific suppression, surface accessibility measurements, and interactional disulfide mapping. The proximal signaling domain is proposed to be a functionally important substructure that converts the rotational signal from the HAMP domain into a displacement of the signaling domain. Clarifying the structural relationships of the Aer domains will guide the design of experiments to determine the signaling pathway. Specific Aim 2 will investigate the aerotaxis signaling mechanism within an Aer homodimer. Reduction of FAD in the Aer PAS domain is proposed to cause a conformational change that induces rotation of a four-helix HAMP bundle, converting the Aer signal output from the kinase-off state to the kinase-on state. Key residues in the FAD-binding cleft will be identified through in silico analyses, site-specific and random mutagenesis, covalent labeling of residues near the isoalloxazine redox center of FAD (using photolabeled-FAD), and FAD-binding measurements. To clarify the sequence of the signaling pathway, successful intragenic complementation studies will be extended to determine whether mutant PAS domains that are locked in the "on" state, signal through the proximal or cognate HAMP-AS-2 helix, or both. We will also use disulfide cross-linking to determine whether the HAMP domain maintains a stable four-helix bundle in both the on and off signaling state. Together these studies should reveal the critical residues of the signaling pathway. Specific Aim 3 will determine the signal(s) sensed by the Aer PAS domain. Although aerotaxis requires the electron transport system, flavin reductase (Fre) reduces Aer-FAD in vitro. A reconstituted aerotaxis system will be used to investigate the role of Fre and test the hypothesis that Aer can sense cytoplasmic redox potential and NADH/NAD ratios, independent of the electron transport system. Elucidating the signaling mechanism in Aer should provide insight into the sensory mechanisms of medically important PAS and HAMP proteins. Investigations of oxygen sensing in E. coli bacteria identified a PAS domain as the sensory module in the receptor. PAS domains are found in 10,000 proteins from bacteria to humans, including medically important proteins. This study seeks to understand how sensory receptors function. The knowledge gained will help scientists understand how more complex sensory systems monitor oxygen concentration and energy levels in cells like human nerve cells that are easily damaged if oxygen is not maintained at adequate levels.