Transhydrogenase: Structure, Dynamics, and Mechanism

转氢酶：结构、动力学和机制

• 批准号: 9035404
• 负责人: Qinghai Zhang
• 金额: $ 36.58万
• 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/9035404
• 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; 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; Proton Pump; Proton-Motive Force; Protons; Reactive Oxygen Species; Reducing Agents; Resolution; Resources; Respiration; Roentgen Rays; Role; Signal Transduction; Site-Directed Mutagenesis; Structure; Structure of beta Cell of islet; System; Thermus thermophilus; Time; design and construction; dimer; electron density; electron diffraction; gene product; 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）偶联到NADPH的形成。在缺乏NADPH的情况下，活性氧（ROS）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，由TH突变引起的氧化应激与2型糖尿病相关。为了了解ROS、线粒体功能障碍与疾病之间的关系，有必要了解TH偶联pmf与NADPH形成的机制。广泛的生化和遗传数据是可用的，但要确定整个酶的晶体结构及其组成亚基的机制是必要的。此外，还需要了解氢化物转移和质子泵送过程中亚基的分布。一种可行的策略是利用嗜热热菌的TH进行酶及其单个组分的结晶。在脂质立方相（LCP）中获得了膜插层结构域的衍射质量晶体；通过结构设计和共结晶，还将获得与可溶性NADP(H)结合结构域（结构域III）配合物的膜结构域的结构。已确定了可溶NAD(H)亚基单独和与结构域III配合物的高分辨结构。完整TH的多种形态的大棱柱形晶体可用于衍射实验。使用低分辨率x射线数据和EM方法（单粒子平均和电子衍射）将组件晶体结构定位在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，将确定复合物内结构域III的运动。结构导向的定点诱变和生化分析（循环和反向氢化物转移，矢量质子泵送）将定义残基功能。对结构、构象状态和功能的了解将有助于推导出质子易位和NADPH形成耦合的机制。