Sensory Transduction in Bacteria

描述(申请人提供)：嗜氧性，即大肠杆菌对氧气的行为反应，是由Aer和TSR受体介导的。Aer是一种内部受体，定位于细胞膜的中部到内侧。N-末端的PAS结构域有一个FAD辅因子，感觉细胞质中的氧化还原和能量水平。Aer的羧基部分也是细胞质的，含有一个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的信号转导机制将为PAS和HAMP结构域之间的信号转导提供第一个深入的研究。PAS和HAMP结构域是重要的感觉模块，在生命系统中广泛存在，但其他研究人员尚未对其进行广泛研究。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结构域相连，形成Aer的输入/输出模块。HAMP结构域的结构将通过体内的二硫键交联和与F.W.Dahlquist合作的核磁共振溶液结构来确定。PAS和HAMP结构域之间的接触面将使用遗传分析来确定，包括等位基因特异性抑制、表面可及性测量和相互作用的二硫键作图。近端的信号域被认为是一个重要的功能亚结构，它将来自HAMP结构域的旋转信号转换为信号域的位移。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同源二聚体内的趋气性信号机制。Aer Pas结构域中FAD的减少被认为是为了引起构象变化，导致四螺旋HAMP束的旋转，将Aer信号从激活关闭状态转换到激活状态。FAD结合裂解中的关键残基将通过计算机分析、定点和随机突变、FAD异四氧嘧啶氧化还原中心附近残基的共价标记(使用光标记FAD)以及FAD结合测量来确定。为了弄清信号通路的序列，成功的基因内互补研究将被扩展到确定锁定在“开”状态的突变的PAS结构域是通过近端或同源的HAMP-AS-2螺旋发出信号，还是两者兼而有之。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都保持稳定的四螺旋束。总而言之，这些研究应该揭示信号通路的关键残基。具体目标3将确定由Aer PAS域感测到的信号(S)。虽然趋气性需要电子传递系统，但黄素还原酶(FRE)在体外可以减少Aer-FAD。重组的趋气性系统将被用来研究Fre的作用，并检验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中的信号机制将有助于深入了解具有重要医学意义的PAS和HAMP蛋白的感觉机制。对大肠杆菌中氧感应的研究发现，PAS结构域是受体中的感觉模块。PAS结构域存在于从细菌到人类的10,000种蛋白质中，包括具有重要医学意义的蛋白质。这项研究试图了解感官受体是如何发挥作用的。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控细胞中的氧气浓度和能量水平，如果氧气没有保持在足够的水平，这些细胞很容易受到损害。

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.