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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.

摘要修饰依赖性限制性系统（MDR）识别并切割修饰的外源DNA。蛋白质被认为在建立细菌基因组的表观遗传景观中起作用，尤其重要的是防止捕食性噬菌体病毒，其中许多包含在它们的DNA中修饰碱基以逃避其他防御系统的检测。大多数抗生素耐药菌，包括耐甲氧西林金黄色葡萄球菌（MRSA）、梭菌艰难梭菌和碳青霉烯耐药肠杆菌科，如肺炎克雷伯菌，无真核同源物存在，使它们成为药物设计的有希望的靶点。抑制这些系统有可能增强噬菌体介导的细菌杀伤的功效，从而提供了新的治疗策略，持续的、耐抗生素的微生物感染。研究基础生物学和 MDRs的机制，并使用这些知识来改善目前的噬菌体治疗方法。检查了McrBC限制系统的结构和功能，这是一个双组分MDR，大肠杆菌McrB含有一个N-末端DNA结合结构域和一个C-末端DNA结合结构域，末端AAA+马达结构域，其水解GTP并介导核苷酸依赖性寡聚化。 McrB的基础GT酶活性通过与其伴侣内切核酸酶McrC相互作用而被刺激。研究提出了一种DNA切割模型，其中McrB和McrC在两个距离处组装在一起，甲基化位点并以依赖于刺激的GTP水解的方式移位。这些的碰撞 McrBC组装触发两条DNA链的切割。 McrBC功能的机制细节仍然不清楚。在目标1中，我们将剖析物种-- 用X射线晶体学研究不同McrB同系物中DNA结合的特异性决定簇，我们还将产生嵌合体，其在不同的生物化学之间交换DNA结合结构域。 McrB同源物，以检验McrBC中的核心水解和裂解机制是在目标2中，我们将使用诱变和动力学测定，以确定负责McrC-β刺激的关键催化组分，在目标3中，我们将确定McrBC的结构和体系结构组织通过X射线晶体学和低温电子显微镜以原子分辨率观察限制复合物。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。