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（由适用提供）：虽然自动数据采集方法已实现了调节途径的描述，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。该提案的总体目标是在分子水平上推导控制整个信号通路的机制，该机制是模型革兰氏阳性细菌中氮调节回路的机制。 In B. subtilis, the metabolism and assimilation of nitrogen is controlled by an unusual network of proteins, distinct from that used by Gram-negative bacteria, that includes the central enzyme of nitrogen metabolism, glutamine synthetase (GS), the global transcription regulators, TnrA and GlnR, and the ammonium transport regulator, GlnK. GS合成谷氨酰胺（Q），这是枯草芽孢杆菌中首选的氮源，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不仅在氮同化中起着核心作用，而且在其谷氨酰胺反馈抑制的形式（GS-Q）中直接与TNRA和GLNR相互作用，也发挥了转录调控。在超过氮期间，形成GS-Q并结合TNRA，以防止该全局活化剂的DNA结合活性，而它通过未知的陪同机制激活GLNR的阻遏活性。在氮条件下，GLNK与TNRA相互作用以促进其结合DNA的能力。这就是枯草芽孢杆菌氮同化途径高度相互联系，最终允许枯草芽孢杆菌检测细胞内氮水平并传输该信号以实现ENZME活性和基因调节。迄今为止，我们已经确定了原子水平枯草芽孢杆菌GS的酶促机制，表明其催化活性和调节在GS蛋白之间是不同的。该项目的目标是进行生化，结构和体内研究，以剖析控制这种氮同化途径的调节机制。这两个组合特定的目的如下：（1）推断出控制GLNR调节网络在内的分子机制，包括GLNR DNA结合，其自身抑制和GS的独特链酮功能。（2）阐明了TNRA DNA结合机制及其通过GS-Q抑制而激活的TNRA DNA结合机制。值得注意的是，GS是已建立的抗菌药物靶标。这，本提案中获得的详细结构信息将提供有关改善药物开发的洞察力，并提供新的靶标，例如TNRA和GLNR，以设计高度特异性的抗菌化学治疗药。