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，固定在细胞质膜的中间。n端PAS结构域具有FAD辅助因子，可感知细胞质中的氧化还原和能量水平。Aer的羧基一半，同样是细胞质，具有一个HAMP结构域和一个在趋化受体中高度保守的信号结构域。阐明Aer信号传导机制将为PAS和HAMP结构域之间的信号转导提供首次深入研究：PAS和HAMP结构域是在生命系统中广泛存在的重要感觉模块，但尚未被其他研究者广泛研究。特异性目的1将研究Aer同二聚体结构域的结构关系。基于结构模型，提出了HAMP结构域是一个四螺旋束，直接与PAS结构域接口，形成Aer的输入/输出模块。HAMP结构域的结构将由体内二硫交联和与Dahlquist合作的核磁共振溶液结构确定。PAS和HAMP结构域之间的接触面将通过遗传分析确定，包括等位基因特异性抑制、表面可达性测量和相互作用二硫图谱。近端信号域被认为是一个功能重要的子结构，它将来自HAMP域的旋转信号转换为信号域的位移。澄清Aer结构域的结构关系将指导实验设计以确定信号通路。特异性目标2将研究Aer同型二聚体中的趋氧信号机制。Aer PAS结构域中FAD的减少引起构象变化，引起四螺旋HAMP束的旋转，将Aer信号输出从激酶关闭状态转换为激酶打开状态。FAD结合间隙中的关键残基将通过硅分析、位点特异性和随机诱变、FAD异alloxazine氧化还原中心附近残基的共价标记（使用光标记-FAD）和FAD结合测量来确定。为了明确信号通路的序列，将扩展成功的基因内互补研究，以确定锁定在“开”状态的突变PAS结构域是通过近端或同源的hmp - as -2螺旋发出信号，还是两者兼有。我们还将使用二硫交联来确定HAMP结构域在打开和关闭信号状态下是否保持稳定的四螺旋束。这些研究共同揭示了信号通路的关键残基。特定的Aim 3将确定由Aer PAS域感知的信号。虽然趋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。重建的可氧性系统将用于研究Fre的作用，并验证Aer可以感知细胞质氧化还原电位和NADH/NAD比率的假设，而不依赖于电子传递系统。阐明Aer中的信号传导机制将有助于深入了解医学上重要的PAS和HAMP蛋白的感觉机制。对大肠杆菌氧感应的研究发现了一个PAS结构域作为受体的感觉模块。PAS结构域存在于从细菌到人类的10,000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是如何监测细胞中的氧气浓度和能量水平的，比如人类神经细胞，如果氧气没有保持在足够的水平，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.