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: 9118245
• 负责人: Maria Schumacher
• 金额: $ 30.93万
• 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/9118245
• 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; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Health; Human; In Vitro; Intervention; Life; Macronutrients Nutrition; Mediating; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Molecular Chaperones; Mutagenesis; N-terminal; Nitrogen; Nitrogen Fixation Genes; Organism; 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

 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)：虽然自动数据采集方法已经能够描述调控途径，但驱动和协调这些过程的详细分子机制仍然未知或不完全表征。这项提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，即模式革兰氏阳性细菌枯草杆菌中氮调节电路的机制。在枯草芽孢杆菌中，氮的代谢和同化是由一个不同于革兰氏阴性菌所使用的unusua蛋白质网络控制的，该网络包括氮代谢的中心酶谷氨酰胺合成酶(GS)、全球转录调节因子TnrA和GlnR以及氨运输调节因子GlnK。GS合成谷氨酰胺(Q)，这是枯草杆菌的首选氮源，而GInR和TnrA调节所有参与氮代谢的蛋白质编码基因的转录。在枯草芽孢杆菌中，GS不仅在氮同化中起核心作用，而且通过与TnrA和GlnR以谷氨酰胺反馈抑制形式(GS-Q)直接相互作用而参与转录调控。在氮过量时，GS-Q形成并与TnrA结合以阻止这种全局激活剂的DNA结合活性，而它通过一种未知的伴侣机制激活GlnR的阻遏活性。在缺氮条件下，GlnK与TnrA相互作用，促进其结合DNA的能力。因此，枯草杆菌的氮同化途径是高度相互联系的，最终使枯草杆菌能够检测到细胞内的氮水平，并传递这一信号来影响酶活性和基因调控。到目前为止，我们已经在原子水平上确定了枯草杆菌GS的酶机制，揭示了其催化活性和调控在GS蛋白中的不同。该项目的目标是进行生化、结构和体内研究，以剖析控制这一氮同化途径的调节机制。这两个多部分的特异性目标如下：(1)推测控制GlnR调控网络的分子机制，包括GlnR与DNA的结合，其自身抑制的调节，以及GS独特的伴侣功能。(2)阐明TnrA DNA结合机制及GlnK的激活和GS-Q的抑制作用。值得注意的是，GS是一个既定的抗菌药物靶点。因此，在本提案中获得的详细结构信息将 为改进的药物开发提供洞察力，并为设计高度特异的抗菌化疗药物提供新的靶点，如TnrA和GlnR。