Transhydrogenase: Structure, Dynamics, and Mechanism

描述(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)偶联呼吸产生的质子动力(PMF)形成NADPH。在NADPH缺乏的情况下，活性氧(ROS)导致线粒体功能障碍，与神经退行性疾病密切相关。在胰岛β细胞中，由于控制胰岛素分泌的氧化还原信号的丢失，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形成耦合的机制。可以获得大量的生化和遗传数据，但要确定一种机制，需要整个酶及其组成亚基的晶体结构。此外，还需要了解氢化物转移和质子泵送过程中亚基的处置情况。一种可行的策略是使用嗜热嗜热菌的TH来结晶酶及其单独的成分。在脂质立方相(LCP)中得到了具有衍射性的膜插层结构域(LCP)晶体；通过结构设计和共结晶，还将得到膜结构域与可溶的NADP(H)结合结构域(结构域III)。解决了可溶性NAD(H)亚基单独的和与结构域III形成的复合体的高分辨结构。具有多种形貌的完整的大棱柱晶体可用于衍射实验。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)，将组分晶体结构定位在Th电子密度包层内，首次揭示了Th的完整结构。在NAD(H)和NADP(H)存在下的SAXS实验，连同晶体结构，将识别复合体中结构域III的运动。结构导向的定点突变和生化分析(循环和反向氢化物转移、矢量质子泵)将定义残基功能。对结构、构象状态和功能的了解将使我们能够推断出耦合质子转移和NADPH形成的机制。

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.