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方面发挥着重要作用 耐药性，在调节人类微生物组方面，它们作为纳米治疗药物具有巨大的潜力。 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的潜力 代理需要对病毒的发展有基本的了解。基因组包装途径 在大型双链 DNA (dsDNA) 病毒中高度保守，无论是原核病毒还是 真核的。在这一大类病毒中，终止酶负责 (i) 切除 来自串联底物的个体基因组（基因组成熟）和（ii）DNA易位到 原衣壳壳（基因组包装）。这些功能由终止酶催化 组装成离散的成熟和包装运动复合体。终止酶由 催化亚基和 DNA 识别亚基，这两者对于基因组包装至关重要 体内。结构和单分子研究为包装运动复合体提供了见解 由分离的催化亚基组成；然而，关于运动复合体的信息很少 含有两个必需的亚基。此外，缺乏关于平等的结构信息 必需的成熟复合物前体。这在一定程度上是由于缺乏良好的特征 全酶制剂和缺乏体外测定来全面评估该途径。我们 已开发出严格的检测方法，其中的生化、生物物理和结构特征 lambda 基因组包装途径可以非常详细地定义。使用这些工具，我们建议 表征结构（冷冻电子显微镜）和功能（生物物理、动力学）特征 成熟复合物，其机制与四聚体类型 IIE/F 限制相似 核酸内切酶。我们直接解决与 DNA 架构相关的新出现的争议 介导复合体稳定性的成熟复合体。接下来我们测试 lambda 马达的假设 也作为四聚体复合物发挥作用，并且马达的 ATP 水解具有很强的协同性； 这些特征与当前公认的范式有很大的不同。最后，我们 描述了一种假定的“核苷酸开关”机制，该机制控制从稳定状态的转变 成熟复合体到与衣壳结合的动态运动复合体，我们严格定义 移位电机的能量预算。拟议的研究将提供结构和机械 两个连续包装复合物及其通过基因组包装的转变的详细信息 途径。这些特征是所有包装基因组的 dsDNA 病毒所共有的。 多联体前体（噬菌体、疱疹）及其结果将具有广泛和普遍的意义。