Complete atomic dissection of the B. subtilis nitrogen regulatory pathway

枯草芽孢杆菌氮调节途径的完整原子解剖

• 批准号: 9313913
• 负责人: Maria Schumacher
• 金额: $ 30.92万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9313913
• 关键词: Ammonium; Anti-Bacterial Agents; Assimilations; Automobile Driving; Bacillus subtilis; Binding; Biochemical; Biological Assay; Biological Availability; C-terminal; Complex; Crystallography; DNA; DNA Binding; DNA Binding Domain; Development; Dissection; Drug Design; Drug Targeting; Enzymes; Family; Feedback; Fluorescence; Gene Expression Regulation; Genes; Genetic Transcription; Glean; Glutamate-Ammonia Ligase; Glutamine; Glutamine-tRNA ligase; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Human; In Vitro; Intervention; Life; Macronutrients Nutrition; Mediating; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Molecular Chaperones; Mutagenesis; N-terminal; Nitrogen; Nitrogen Fixation Genes; Organism; Pathogenicity; Pathway interactions; Play; Process; Proteins; Regulation; Regulatory Pathway; Role; Signal Pathway; Signal Transduction; Site; Source; Structural Models; Structure; System; Tail; Transcriptional Regulation; Work; biological systems; data acquisition; design; drug development; improved; in vivo; inhibitor/antagonist; insight; nitrogen metabolism; novel; prevent; protein structure; public health relevance

 DESCRIPTION (provided by applicant): While automated data acquisition methods have enabled the delineation of regulatory pathways, the detailed molecular mechanisms that drive and coordinate these processes remain unknown or incompletely characterized. The overall goal of this proposal is to deduce, at the molecular level, the mechanisms that control an entire signaling pathway, that of the nitrogen regulatory circuit in the model Gram-positive bacterium B. subtilis. In B. subtilis, the metabolism and assimilation of nitrogen is controlled by an unusua network of proteins, distinct from that used by Gram-negative bacteria, that include the central enzyme of nitrogen metabolism, glutamine synthetase (GS), the global transcription regulators, TnrA and GlnR, and the ammonium transport regulator, GlnK. GS synthesizes glutamine (Q), which is the preferred nitrogen source in B. subtilis, while GInR and TnrA regulate the transcription of all protein-encoding genes involved in nitrogen metabolism. In B. subtilis, GS plays a central role not only in nitrogen assimilation, but also transcription regulation by interacting directly with TnrA and GlnR in its glutamine feedback-inhibited form (GS-Q). During nitrogen excess, GS-Q is formed and binds TnrA to prevent the DNA binding activity of this global activator, while it activates the repressor activity of GlnR by an unknown chaperoning mechanism. During nitrogen poor conditions, GlnK interacts with TnrA to facilitate its ability to bind DNA. Thus, the B. subtilis nitrogen assimilation pathway is highly interconnected, ultimately allowing B. subtilis to detect intracellular nitrogen levels and transmit this signal to effect enzme activity and gene regulation. To date, we have determined the enzymatic mechanism of B. subtilis GS at the atomic level, revealing that its catalytic activity and regulation are distinct among GS proteins. The goals of this project are to perform biochemical, structural and in vivo studies to dissect the regulatory mechanisms that control this nitrogen assimilation pathway. The two, multi-part Specific Aims are as follows: (1) Deduce the molecular mechanisms controlling the GlnR regulatory network including GlnR DNA-binding, its regulation by autoinhibition, and the unique chaperone function of GS. (2) Elucidate the TnrA DNA binding mechanism and its activation by GlnK and inhibition by GS-Q. Notably, GS is an established antibacterial drug target. Thus, the detailed structural information obtained in this proposal will provide insight into improved drug development as well as provide new targets, such as TnrA and GlnR, for the design of highly specific, antibacterial chemotherapeutics.

 描述（由申请人提供）：虽然自动化数据采集方法已经能够描绘调控途径，但驱动和协调这些过程的详细分子机制仍然未知或未完全表征。该建议的总体目标是在分子水平上推断控制整个信号通路的机制，即革兰氏阳性菌B模型中的氮调节回路。枯草杆菌。在B。在枯草杆菌中，氮的代谢和同化由独特的蛋白质网络控制，与革兰氏阴性细菌所使用的蛋白质不同，其包括氮代谢的中心酶谷氨酰胺合成酶（GS）、全局转录调节因子TnrA和GlnR以及铵转运调节因子GlnK。GS合成谷氨酰胺（Q），其是B中的优选氮源。枯草杆菌，而GInR和TnrA调节参与氮代谢的所有蛋白质编码基因的转录。在B。在枯草芽孢杆菌中，GS不仅在氮同化中起重要作用，而且通过以其谷氨酰胺反馈抑制形式（GS-Q）直接与TnrA和GlnR相互作用而在转录调节中起重要作用。在氮过量期间，GS-Q形成并结合TnrA以阻止该全局激活剂的DNA结合活性，同时其通过未知的伴侣机制激活GlnR的阻遏物活性。在氮缺乏条件下，GlnK与TnrA相互作用以促进其结合DNA的能力。因此，B.枯草杆菌氮同化途径是高度相互关联的，最终允许B。枯草芽孢杆菌的细胞内氮水平的检测和传递这一信号，影响酶的活性和基因调控。到目前为止，我们已经确定了酶的机制B。在原子水平上对枯草芽孢杆菌GS的催化活性进行了研究，揭示了其催化活性和调节在GS蛋白中的不同。该项目的目标是进行生物化学，结构和体内研究，以剖析控制这一氮同化途径的调节机制。（1）推导GlnR调控网络的分子机制，包括GlnR与DNA的结合、自身抑制的调控和GS独特的分子伴侣功能。(2)阐明TnrA DNA结合机制及GlnK对TnrA DNA结合的激活作用和GS-Q对TnrA DNA结合的抑制作用。值得注意的是，GS是一个既定的抗菌药物的目标。因此，本建议书中获得的详细结构信息将 为改进药物开发提供见解，并为设计高度特异性的抗菌化疗药物提供新的靶点，如TnrA和GlnR。