Structure and Function of Essential Nucleoprotein ComplexesAlong a Viral Genome Packaging Pathway

病毒基因组包装途径中必需核蛋白复合物的结构和功能

• 批准号: 9920164
• 负责人: Carlos Enrique Catalano
• 金额: $ 37万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920164
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Antibiotic Resistance; Antiviral Agents; Architecture; Bacteriophage lambda; Bacteriophages; Behavior; Binding; Biochemical; Biological; Biological Assay; Biophysics; Budgets; Capsid; Catalytic Domain; Chemicals; Complex; Coupling; Cryoelectron Microscopy; DNA; DNA Restriction Enzymes; Development; Double Stranded DNA Virus; Electron Microscopy; Enzymes; Evolution; Excision; Genome; Goals; Health; Holoenzymes; Human; Human Microbiome; Individual; Kinetics; Mediating; Modeling; Molecular; Motor; Nucleoproteins; Nucleotides; Pathogenicity; Pathway interactions; Play; Preparation; Process; Reaction; Research; Role; Site; Structure; System; Testing; Therapeutic; Time; Ursidae Family; Viral; Viral Genome; Viral Packaging; Virus; Virus Assembly; Work; design; experience; experimental study; human disease; in vitro Assay; in vivo; insight; monomer; multitask; nanotherapeutic; novel; programs; single molecule; stoichiometry; terminase; theranostics; tool; viral DNA; virtual; virus development

Project Summary. Bacteriophages play a major role in bacterial evolution, in mediating bacterial pathogenicity and antibiotic resistance, in modulating the human microbiome and they have great potential as nanotherapeutics. Understanding these issues with respect to human disease and harnessing their potential as theranostic agents requires a fundamental understanding of virus development. The genome packaging pathways are strongly conserved in the large double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic. In this broad class of viruses, a terminase enzyme is responsible for (i) excision of an individual genome from concatemeric substrate (genome maturation) and (ii) translocation of DNA into a procapsid shell (genome packaging). These functions are catalyzed by terminase enzymes assembled into discrete maturation and packaging motor complexes. Terminases are composed of a catalytic subunit and a DNA recognition subunit, both of which are essential for genome packaging in vivo. Structural and single-molecule studies have provided insight into packaging motor complexes composed of the catalytic subunit in isolation; however, there is little information on motor complexes containing both essential subunits. Further, there is a dearth of structural information on the equally essential maturation complex precursor. This is due, in part, to the absence of well-characterized holoenzyme preparations and a dearth of in vitro assays to comprehensively assess the pathway. We have developed rigorous assays in which the biochemical, biophysical and structural features of the lambda genome-packaging pathway can be defined in great detail. Using these tools, we propose to characterize the structural (cryo-electron microscopy) and functional (biophysical, kinetic) features of the maturation complex, which show mechanistic similarity to the tetrameric type IIE/F restriction endonucleases. We directly address an emerging controversy relating to the DNA architecture in the maturation complex that mediates complex stability. We next test the hypothesis that the lambda motor also functions as a tetrameric complex and that ATP hydrolysis by the motor is strongly cooperative; these features represent a significant departure from currently accepted paradigms. Finally, we characterize a putative "nucleotide switch" mechanism that controls the transition from the stable maturation complex to the dynamic motor complex bound to the capsid and we rigorously define the energy budget of the translocating motor. The proposed studies will provide structural and mechanistic detail on two sequential packaging complexes and their transition through the genome-packaging pathway. These features are shared by all of the dsDNA viruses that package genomes from concatemeric precursors (phage, herpes) and the results will be of broad and general significance.

项目摘要。 噬菌体在细菌进化、介导细菌致病性和抗生素等方面发挥着重要作用 耐药性，在调节人类微生物组中，它们具有作为纳米治疗剂的巨大潜力。 了解这些与人类疾病有关的问题，并利用它们作为治疗诊断学的潜力 病原体需要对病毒发展有基本的了解。基因组包装途径 在大双链DNA（dsDNA）病毒中是高度保守的，包括原核和 真核生物在这一广泛类别的病毒中，末端酶负责（i）切除病毒的末端， 单个基因组从多联体底物（基因组成熟）和（ii）DNA易位到 原衣壳壳（基因组包装）。这些功能由末端酶催化 组装成离散的成熟和包装运动复合体。终端由一个 催化亚基和DNA识别亚基，这两个亚基对于基因组包装都是必不可少的。 vivo.结构和单分子的研究提供了深入了解包装电机复合物 由催化亚基组成的隔离;然而，很少有关于马达复合体的信息 含有两种基本亚基。此外，同样缺乏结构信息， 必需成熟复合物前体这部分是由于缺乏良好的特征， 全酶制剂和缺乏体外试验来全面评估该途径。我们 已经开发了严格的分析，其中的生物化学，生物物理和结构特征的 λ基因组包装途径可以更详细地定义。利用这些工具，我们建议 表征的结构（冷冻电子显微镜）和功能（生物物理，动力学）的特点， 成熟复合物，其显示与四聚体IIE/F型限制的机制相似性 核酸内切酶。我们直接解决一个新出现的争议有关的DNA架构在 成熟复合体，介导复合体的稳定性。我们接下来测试的假设， 也作为四聚体复合物起作用，并且由马达引起的ATP水解是强烈合作的; 这些特征代表了与当前接受的范例的显著偏离。最后我们 描述了一种假定的“核苷酸开关”机制，该机制控制从稳定的 成熟复合物与结合到衣壳的动态运动复合物结合，并且我们严格定义了 移位马达的能量预算。拟议的研究将提供结构和机制 详细介绍了两个顺序包装复合物及其通过基因组包装的过渡 通路这些特征是所有dsDNA病毒所共有的，这些dsDNA病毒包装来自 多联体前体（噬菌体、疱疹），并且结果将具有广泛和普遍意义。