Structural and Functional Characterization of the McrBC Restriction System

McrBC 限制系统的结构和功能表征

基本信息

批准号：
10318156
负责人：
Joshua S Chappie
金额：
$ 31.71万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318156
关键词：
Address Architecture Atomic Resolution X-Ray Crystallography Bacteria Bacterial Antibiotic Resistance Bacterial Genome Bacterial Infections Bacteriophages Binding Biochemical Biochemistry Biological Biological Assay Biological Models Biology C-terminal Cessation of life Chimera organism Clostridium difficile Complex Conflict (Psychology)Coupled Cryoelectron Microscopy Cytosine DNA DNA Binding DNA Binding Domain Detection Distant Drug Design Drug Targeting Engineering Enhancers Epigenetic Process Escherichia coli Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Health Helicobacter pylori Homologous Gene Human Hydrolysis Infection Kinetics Klebsiella pneumoniae Knowledge Lead Lytic Mediating Microbial Antibiotic Resistance Modeling Modification Molecular Molecular Conformation Motor Multi-Drug Resistance Mutagenesis N-terminal Nature Nucleotides Pathway interactions Play Proteins Regulation Research Resolution Role Side Site Structure Substrate Specificity Superbug System Testing Therapeutic Agents Thermococcus United States Virus X-Ray Crystallography base carbapenem-resistant Enterobacteriaceae clinically relevant combat design endonuclease improved inhibitor insight interest methicillin resistant Staphylococcus aureus novel therapeutic intervention novel therapeutics nuclease pressure stoichiometry tool

项目摘要

Abstract Modification-dependent restriction systems (MDRs) recognize and cleave modified foreign DNA. These proteins are thought to play a role in establishing the epigenetic landscape of bacterial genomes and are especially important in protecting against predatory bacteriophage viruses, many of which incorporate modified bases into their DNA to evade detection by other defense systems. While MDRs can be found in most antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, and carbapenem-resistant enterobacteriaceae like Klebsiella pneumoniae, no eukaryotic homologs exist, making them promising targets for drug design. Inhibiting these systems has the potential to enhance the efficacy of phage-mediated bacterial killing, thus providing new therapeutic strategies to combat persistent, antibiotic resistant microbial infections. It is our long-term goal to study the basic biology and mechanisms of MDRs and use this knowledge to improve current phage therapy approaches. This proposal examines the structure and function of the McrBC restriction system, a two-component MDR that targets DNA containing methylated cytosines. E. coli McrB contains an N-terminal DNA binding domain and a C- terminal AAA+ motor domain that hydrolyzes GTP and mediates nucleotide-dependent oligomerization. McrB’s basal GTPase activity is stimulated via interaction with its partner endonuclease McrC. Biochemical studies suggest a model for DNA cleavage in which McrB and McrC assemble together at two distant methylated sites and translocate in a manner dependent on stimulated GTP hydrolysis. Collision of these McrBC assemblies triggers cleavage of both DNA strands. Despite this model, the molecular and mechanistic details underlying McrBC function remain poorly defined. In Aim 1, we will dissect the species- specific determinants of DNA binding in different McrB homologs using X-ray crystallography and biochemistry. We will also generate chimeras that exchange the DNA binding domains between different McrB homologs to test the hypothesis that the core hydrolysis and cleavage machineries in McrBC are conserved and have adapted to different evolutionary pressures via a modular design. In Aim 2, we will use mutagenesis and kinetic assays to identify the critical catalytic components responsible for McrC-stimulated GTPase activity. In Aim 3, we will determine the structure and architectural organization of the McrBC restriction complex at atomic resolution by X-ray crystallography and cryo-electron microscopy. These efforts will provide new insights into how McrBC complexes bind DNA, assemble, and hydrolyze GTP.

抽象的识别并清除修改的外国DNA的修改依赖性限制系统（MDR）。这些人们认为蛋白质在建立细菌基因组的表观遗传景观方面发挥作用，并且是在防止掠食性细菌病毒中尤其重要，其中许多都合并修改后的碱基将其他防御系统视为检测到DNA。虽然可以在大多数抗生素耐药细菌，包括甲氧西林金黄色葡萄球菌（MRSA），梭状芽胞杆菌艰难梭菌和耐碳青霉烯类肠杆菌科，如肺炎，无真核同源物存在，成为药物设计的有希望的目标。抑制这些系统有可能增强噬菌体介导的细菌杀死的有效性，从而提供了新的治疗策略来对抗持续的，抗生素耐药的微生物感染。研究基本生物学和 MDR的机制，并使用这些知识来改善当前的噬菌体疗法。这个建议检查MCRBC限制系统的结构和功能，这是一个针对的两个组件MDR 含有甲基化胞嘧啶的DNA。大肠杆菌MCRB包含一个N末端DNA结合结构域和c- 末端AAA+运动结构域水解GTP并介导核苷酸依赖性寡聚。 MCRB的基础GTPase活性是通过与其伴侣核酸内切酶MCRC的相互作用刺激的。生化研究提出了一个DNA裂解的模型，其中MCRB和MCRC在两个遥远的甲基化位点和以某种方式转运的方式取决于刺激的GTP水解。这些碰撞 MCRBC组件触发了两个DNA链的裂解。尽管这种模型，分子和 MCRBC功能基础的机械细节仍然很差。在AIM 1中，我们将剖析物种 - 使用X射线晶体学和生物化学。我们还将生成在不同的嵌合体之间交换DNA结合域之间的嵌合体 MCRB同源物测试以下假设：MCRBC中的核心水解和裂解机械是保守并通过模块化设计适应了不同的进化压力。在AIM 2中，我们将使用诱变和动力学测定，以识别负责MCRC刺激的关键催化成分 GTPase活性。在AIM 3中，我们将确定MCRBC的结构和建筑组织 X射线晶体学和冷冻电子显微镜在原子分辨率下进行限制复合物。这些努力将为MCRBC复合物如何结合DNA，组装和水解GTP提供新的见解。