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

基本信息

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
项目类别：
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

项目摘要

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不完全表征，该提案的总体目标是在分子水平上进行推断。，控制整个信号通路的机制，即模型革兰氏阳性细菌枯草芽孢杆菌中的氮调节回路。在枯草芽孢杆菌中，氮的代谢和同化是由一个不寻常的网络控制的。与革兰氏阴性细菌使用的蛋白质不同，这些蛋白质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，合成谷氨酰胺 (Q)。，这是枯草芽孢杆菌中的首选氮源，而 GInR 和 TnrA 则调节枯草芽孢杆菌中所有参与氮代谢的蛋白质编码基因的转录。不仅在氮同化中发挥着核心作用，而且还通过与谷氨酰胺反馈抑制形式 (GS-Q) 的 TnrA 和 GlnR 直接相互作用而在转录调节中发挥核心作用，在氮过量期间，GS-Q 形成并与 TnrA 结合以阻止 DNA。该全局激活剂的结合活性，同时它通过未知的陪伴机制激活 GlnR 的阻遏活性，在氮缺乏条件下，GlnK 与 TnrA 相互作用以促进。因此，枯草芽孢杆菌氮同化途径是高度互连的，最终使枯草芽孢杆菌能够检测细胞内氮水平并传递该信号以影响酶活性和基因调控。迄今为止，我们已经确定了酶促机制。该项目的目标是进行生化、结构和体内研究，以剖析其调节机制。控制该氮同化途径的两个、多部分具体目标如下：（1）推断控制GlnR调节网络的分子机制，包括GlnR DNA结合、其通过自抑制的调节以及GS 2的独特伴侣功能。 ) 阐明 TnrA DNA 结合机制及其通过 GlnK 的激活和 GS-Q 的抑制值得注意的是，GS 是一个已确定的抗菌药物靶标。本提案中获得的结构信息将提供对改进药物开发的深入了解，并为设计高度特异性的抗菌化疗药物提供新的靶点，例如 TnrA 和 GlnR。