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: 8208225
• 负责人: Thomas M Duncan
• 金额: $ 43.1万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208225
• 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; 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

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 (FOF1) 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.

ATP合酶是一种膜结合的能量耦合旋转马达，负责 在动物、植物和许多细菌中合成大多数细胞ATP。它由两个子复合体组成 具有不同的部分功能：FO复合物含有跨膜亚基，在膜中起作用。 质子的运输; F1是一个外周复合物，其中含有催化核苷酸结合 ATP合成的位点。FO和F1通过两个茎状连接的亚基耦合： 中心转子轴和外围定子。在体外，F1可以作为水溶性的 这种酶只催化ATP的净水解，这也是一个简单的子系统， 研究这种酶的许多酶特性。F1和完整催化的一般特征 ATP合酶（FOF 1）在所有类型的酶（线粒体、叶绿体和线粒体）中共享。 细菌），但调节其功能的一些因素似乎是适应或独特的代谢 特定生物体的需求。例如，越来越多的证据表明， C-末端结构域（CTD）的构象变化在调节蛋白质表达中起一种或多种作用。 在细菌和叶绿体的ATP合酶中能量偶联的活性和/或效率。 本项目的长期目标是详细了解E。大肠杆菌ATP合酶 结构、功能和调节，从而提供定量实验模型系统， 了解这个重要酶“家族”的共同结构/功能特征。 为了实现这一目标，我们将把研究重点放在两个方向： 在目标1中，我们将对E. coli F1-ATPase。这 结构工作的目的是提供原子快照的EcF 1-<$在一个扩展的亚基 （开放）和闭合构象。我们假设这两种结构构象， 分别最好地描述了抑制和激活构象所采用的亚基， 是E. coliFOF 1ATP合成酶。 在目标2中，我们将研究亚基对大肠杆菌F1和FOF 1的抑制作用的动态性质。 利用<$突变体和<$中的二硫键交联，我们将限制或偏向可能的构象。 的CTD状态，以便更好地定义<$在EcF 1和EcFOF 1中的调节作用， 的CTD的构象是这些角色所必需的。此外，我们将探讨 使用跨越全部或部分<$-CTD的肽来反式抑制F1或FOF 1的活性。这将 为选择性抑制细菌ATP酶的小分子设计提供合理依据 而不是线粒体酶

项目成果

Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.

通过在亚基 δ 中引入拟磷突变，改进了大肠杆菌 ATP 合酶催化复合物 (F1) 的结晶。

• DOI: 10.1107/s1744309112036718
• 发表时间: 2012
• 期刊: Acta crystallographica. Section F, Structural biology and crystallization communications
• 作者: Roy,Ankoor;Hutcheon,MarcusL;Duncan,ThomasM;Cingolani,Gino
• 通讯作者: Cingolani,Gino

Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation.

• DOI: 10.1038/nsmb.2058
• 发表时间: 2011-06
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Cingolani, Gino;Duncan, Thomas M.
• 通讯作者: Duncan, Thomas M.

Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase.

当从 ATP 合酶的 ϵ 亚基中删除 5 个 C 末端残基时，大肠杆菌的有氧生长会减少，并且 ATP 合成会被选择性地抑制。

• DOI: 10.1074/jbc.m115.665059
• 发表时间: 2015
• 期刊: The Journal of biological chemistry
• 作者: Shah,NamanB;Duncan,ThomasM
• 通讯作者: Duncan,ThomasM