Transhydrogenase: Structure, Dynamics, and Mechanism

转氢酶：结构、动力学和机制

• 批准号: 8629282
• 负责人: Charles David Stout
• 金额: $ 36.01万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8629282
• 关键词: Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Process; C-terminal; Catalysis; Charge; Complex; Coupled; Couples; Coupling; Crystallization; Data; Data Set; Detergents; Electron Microscopy; Electrons; Enzymes; Health; Huntington Disease; Hyperglycemia; Individual; Inner mitochondrial membrane; Insulin Resistance; Knowledge; Lead; Link; Malignant Neoplasms; Maps; Measurement; Membrane; Methods; Microscopy; Mitochondria; Mitochondrial Diseases; Molecular; Molecular Conformation; Morphology; Motion; Movement; Multienzyme Complexes; Mutagenesis; Mutation; NAD(P)+ transhydrogenase; NADH; NADP; Neurodegenerative Disorders; Niacinamide; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Phase; Positioning Attribute; Production; Proteins; Proton Pump; Proton-Motive Force; Protons; Reactive Oxygen Species; Reducing Agents; Resolution; Resources; Respiration; Roentgen Rays; Role; Signal Transduction; Site-Directed Mutagenesis; Solutions; Structure; Structure of beta Cell of islet; System; Thermus thermophilus; Time; design and construction; dimer; electron density; improved; insulin secretion; mitochondrial dysfunction; mutant; particle; reconstruction; research study

DESCRIPTION (provided by applicant): The energy-linked inner mitochondrial membrane enzyme nicotinamide nucleotide transhydrogenase (TH) couples the proton motive force (pmf) generated by respiration to formation of NADPH. In the absence of NADPH reactive oxygen species (ROS) lead to mitochondrial dysfunction which is strongly correlated with neurodegenerative diseases. In pancreatic beta cells, oxidative stress arising from TH mutations is correlated with type 2 diabetes due to loss of the redox signaling that controls insulin secretion. To understand the relationship between ROS, mitochondrial dysfunction and disease, it is necessary to understand the mechanism by which TH couples the pmf with formation of NADPH. Extensive biochemical and genetic data are available, but to define a mechanism crystal structures of the entire enzyme and its constituent subunits are required. Further, knowledge of the dispositions of the subunits during hydride transfer and proton pumping are required. A viable strategy employs TH from Thermus thermophilus for crystallization of the enzyme and its individual components. Diffraction quality crystals of the membrane intercalated domains have been obtained in the lipidic cubic phase (LCP); structures of the membrane domains in complex with the soluble, NADP(H) binding domain (domain III) will also be obtained via construct design and co- crystallization. High resolution structures of the soluble NAD(H) subunit alone and in complex with domain III have been solved. Large prismatic crystals of intact TH in multiple morphologies are available for diffraction experiments. The component crystal structures will be positioned within the TH electron density envelop using lower resolution X-ray data and EM methods (single particle averaging and electron diffraction), revealing for the first time a complete structure of TH. SAXS experiments in the presence of NAD(H) and NADP(H), together with the crystal structures, will identify motions of domain III within the complex. Structure guided site- directed mutagenesis and biochemical assays (cyclic and reverse hydride transfer, vectorial proton pumping) will define residue functions. Knowledge of structure, conformational states, and function will enable a mechanism for coupling proton translocation and NADPH formation to be deduced.

描述（由申请人提供）：能量连接的线粒体内膜酶烟酰胺核苷酸转氢酶（TH）将呼吸产生的质子动力（pmf）耦合到NADPH的形成。在缺乏 NADPH 的情况下，活性氧 (ROS) 会导致线粒体功能障碍，这与神经退行性疾病密切相关。在胰腺 β 细胞中，由于控制胰岛素分泌的氧化还原信号丧失，TH 突变引起的氧化应激与 2 型糖尿病相关。为了了解ROS、线粒体功能障碍和疾病之间的关系，有必要了解TH将pmf与NADPH的形成耦合的机制。可以获得大量的生化和遗传数据，但需要定义整个酶及其组成亚基的晶体结构机制。此外，需要了解氢化物转移和质子泵送期间亚基的配置。一种可行的策略是利用来自嗜热栖热菌的 TH 来结晶酶及其各个组分。在脂质立方相（LCP）中获得了膜插域的衍射质量晶体；与可溶性NADP(H)结合结构域（结构域III）复合的膜结构域的结构也将通过构建体设计和共结晶获得。单独的可溶性 NAD(H) 亚基以及与结构域 III 复合的可溶性 NAD(H) 亚基的高分辨率结构已得到解决。多种形态的完整 TH 的大棱柱晶体可用于衍射实验。组件晶体结构将使用较低分辨率的 X 射线数据和 EM 方法（单粒子平均和电子衍射）定位在 TH 电子密度包络内，首次揭示 TH 的完整结构。在 NAD(H) 和 NADP(H) 存在下的 SAXS 实验以及晶体结构将识别复合物中结构域 III 的运动。结构引导的定点诱变和生化测定（循环和反向氢化物转移、矢量质子泵）将定义残留物功能。了解结构、构象状态和功能将能够推断出质子易位和 NADPH 形成的耦合机制。