Energy Coupling and Regulation in the ATP Synthase of E. coli

大肠杆菌 ATP 合成酶的能量耦合和调控

• 批准号: 7998197
• 负责人: Thomas M Duncan
• 金额: $ 31.54万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7998197
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; Adopted; Animals; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biological Models; C-terminal; Cardiac; Catalysis; Catalytic Domain; Cells; Chloroplasts; Complement; Complex; Coupled; Coupling; Cysteine; Defect; Disulfides; Enzymes; Escherichia coli; Event; Experimental Models; F1-ATPase; Family; Goals; Health; Homologous Gene; Human; In Vitro; Infection; Ischemia; Lead; Membrane; Metabolic; Metabolism; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular Conformation; Motor; Nature; Nucleotides; Organism; Peptides; Peripheral; Plants; Play; Protons; Regulation; Research; Resolution; Role; Structure; System; Water; Work; antibiotic design; base; crosslink; design; disulfide bond; fighting; human disease; insight; mutant; novel strategies; small molecule

DESCRIPTION (provided by applicant): The ATP synthase is a membrane-bound, energy-coupling rotary motor that is responsible for the synthesis of most cellular ATP in animals, plants and many bacteria. It consists of two sub-complexes with distinct, partial functions: the FO complex contains transmembrane subunits and functions in the transport of protons; the F1 is a peripheral complex, which contains the catalytic nucleotide binding sites for ATP synthesis. FO and F1 are coupled through two stalk-like connections of subunits: a central rotor shaft and a peripheral stator. In vitro, F1 can be dissociated from FO as a water-soluble enzyme that only catalyzes net hydrolysis of ATP, and this also serves as a simple sub-system for studying much of the enzymatic features of the enzyme. General features of catalysis by F1 and intact ATP synthase (FO F1) are shared among all types of the enzyme (mitochondrial, chloroplast and bacterial), but some factors that regulate its function appear to be adapted or unique to the metabolic demands of the specific organism. For example, mounting evidence indicates that in E.coli conformational changes in ?'s C-terminal domain (CTD) play one or more roles in regulating the activity and/or efficiency of energy coupling in the ATP synthase of bacteria and chloroplasts. The long-term goal of this project aims to gain a detailed understanding of the E. coli ATP synthase structure, function and regulation and thus provide a quantitative experimental model system to understand common structural/functional features of this important enzyme `family'. To achieve this goal we will focus our research in two directions: In Aim 1, we will carry out high-resolution crystallographic studies on the E. coli F1-ATPase. This structural work is aimed at providing atomic snapshots the EcF1-? with the ? subunit in an extended (open) and closed conformation. We hypothesize that these two structural conformations, respectively, best describe the inhibitory and activating conformations adopted by the ? subunit, which is a critical regulator of E. coli FOF1 ATP synthase. In Aim 2 we will study the dynamic nature of the ? subunit's inhibitory action on E.coli F1 and FOF1. Using ? mutants and disulfide crosslinking within ? we will restrict or bias the possible conformational states of ?'s CTD, in order to better define the regulatory roles of ? in EcF1 and EcFOF1, and which conformations of ?'s CTD are necessary for these roles. In addition, we will explore the possibility of using peptides spanning all or part of the ?-CTD to inhibit, in trans, the activity of F1 or FOF1. This will guide the rational basis for design of small molecules that selectively inhibit bacterial ATP synthases and not the mitochondrial enzyme. PUBLIC HEALTH RELEVANCE: The ATP synthase is a central enzyme in cellular metabolism - it is responsible for making most ATP, the primary energy currency used inside cells. Defects in its assembly or function can cause human diseases and aggravate harmful events such as cardiac ischemia. Understanding unique features of the bacterial ATP synthase may lead to new approaches to fight infections.

描述（由申请人提供）：ATP合酶是一种膜结合的能量耦合旋转马达，负责动物、植物和许多细菌中大多数细胞ATP的合成。它由两个具有不同部分功能的亚复合物组成：FO复合物含有跨膜亚基，并在质子运输中发挥作用; F1是外周复合物，含有ATP合成的催化核苷酸结合位点。FO和F1通过两个子单元的杆状连接件耦合：中心转子轴和外围定子。在体外，F1可以作为一种水溶性酶从FO中解离出来，该酶仅催化ATP的净水解，这也可以作为一个简单的子系统用于研究该酶的许多酶促特征。F1和完整ATP合酶（FO F1）催化的一般特征在所有类型的酶（线粒体，叶绿体和细菌）中共享，但调节其功能的一些因子似乎适应或独特于特定生物体的代谢需求。例如，越来越多的证据表明，在大肠杆菌的构象变化？的C-末端结构域（CTD）在调节细菌和叶绿体的ATP合酶中能量偶联的活性和/或效率中起一种或多种作用。本项目的长期目标是详细了解E。coli ATP合成酶的结构、功能和调控，从而提供了一个定量的实验模型系统，以了解这一重要酶“家族”的共同结构/功能特征。为了实现这一目标，我们将把我们的研究集中在两个方向上：在目标1中，我们将对E. coli F1-ATPase。这一结构工作的目的是提供原子快照的ECF 1-？和那个？亚基处于伸展（开放）和闭合构象。我们假设，这两个结构构象，分别最好地描述了通过的抑制和激活构象？亚基，是E. coliFOF 1ATP合成酶。在目标2，我们将研究的动态性质？亚基对大肠杆菌F1和FOF 1的抑制作用。吸毒？突变体和二硫键交联内？我们将限制或偏向可能的构象状态？的CTD，以更好地定义的监管作用？在EcF 1和EcFOF 1中，哪些构象？的CTD是这些角色所必需的。此外，我们将探索使用肽跨越全部或部分？CTD以反式抑制F1或FOF 1的活性。这将为设计选择性抑制细菌ATP酶而不是线粒体酶的小分子提供合理的基础。公共卫生相关性：ATP合成酶是细胞代谢的中心酶-它负责制造大多数ATP，细胞内使用的主要能量货币。其组装或功能的缺陷可引起人类疾病并加重有害事件，如心脏缺血。了解细菌ATP合酶的独特功能可能会导致对抗感染的新方法。