一类潜在的抗生素药物靶点蛋白NDH-2的结构与功能研究
摘要
电子传递链，又称呼吸链，由五个蛋白质复合物组成，是将代谢过程中产生的电子从NADH向氧分子传递的系统，是生物体最重要的能量来源。其中的复合物I，即NADH：泛醌氧化还原酶，是电子进入电子传递链的入口。在多种低等生物中，复合物I可以被一类称为二型NADH脱氢酶（NDH-2）的蛋白所取代。NDH-2附着在细胞膜或线粒体内膜上，以FAD为辅基，催化电子从NADH传递给泛醌（UQ）。NDH-2在疟原虫、结核杆菌等多种病原微生物中高度保守，而且已被证明是一类潜在的药物靶点。同时，在人体细胞中异源表达Ndi1蛋白（酿酒酵母的NDH-2），被认为可以作为一种基因治疗手段应用于复合物I缺陷引起的多种人类疾病，尤其是帕金森氏病等神经退行性疾病的治疗中。
本文以NDH-2家族的典型代表——酿酒酵母的Ndi1蛋白为研究对象，解析了其高分辨率晶体结构及Ndi1与NADH、UQ以及NADH-UQ三种底物复合物的晶体结构。在所有晶体结构中，Ndi1均以同源二聚体的形式存在。结构分析和序列比对发现，Ndi1具有一个独特的并且在NDH-2家族中序列高度保守的C端结构域（CTD），该结构域介导了Ndi1二聚体化，进而使其形成一个广泛的疏水区域，该区域赋予了Ndi1附着在线粒体内膜上的能力。后续的生物化学和细胞生物学实验都证实了CTD对于Ndi1功能的重要性。同时，三种Ndi1-底物复合物结构的解析揭示了Ndi1与底物的结合方式，并证明了Ndi1中同时存在两个泛醌结合位点。电子顺磁共振实验的结果首次鉴定出在Ndi1介导的电子传递过程中有半醌自由基的出现，同时功率饱和实验支持有两个半醌自由基参与电子传递反应的理论。据此，本论文提出了Ndi1介导电子传递的模型。
本文的研究成果为探讨NDH-2介导电子传递的详细机制、针对NDH-2的药物设计以及更好地将Ndi1应用于基因治疗奠定了良好的基础。本论文综合运用了结构生物学、生物化学与分子生物学、细胞生物学以及物理学等多个学科的实验方法系统地研究了NDH-2家族最具代表性的蛋白Ndi1的多方面特性，全方位地揭示了该蛋白发挥呼吸链电子传递入口这一重要生物学功能的机理。
The electron transport chain, or the respiratory chain, comprising of complexes I-V,transports the electrons derived from metabolic processes from NADH to the oxygenmolecule, and is the most important energy source for living organisms. Complex I, orNADH-ubiquinone oxidoreductase, is thus the entry point for these electrons. In manyorganisms, however, complex I could be replaced by type-II NADH dehydrogenases(NDH-2s), which catalyze the electron transfer from NADH to ubiquinone (UQ), withFAD as a prosthetic group. NDH-2s are highly conserved in many pathogenicmicroorganisms including Mycobacterium tuberculosis and Plasmodium falciparum,and have been recognized as potential drug targets. Meanwhile, heterologous expressionof Ndi1(an NDH-2in Saccharomyces cerevisiae) in human cells has been proposed totreat human diseases caused by complex I defects as typified by Parkinson’s diseases.
In this study, we have solved the high-resolution crystal structures of Ndi1and itsthree types of enzyme-substrate complexes with NADH, UQ and NADH-UQ,respectively. Ndi1formed a homodimer in all the structures. Structural analysis andsequence alignment identified that Ndi1possesses a C-terminal domain (CTD) which ishighly conserved among NDH-2s. The CTD mediates the homodimerization of Ndi1,thus generating an extensive hydrophobic region in the protein to facilitate itsmembrane attachment, which has been confirmed by the subsequent biochemical andcell biological experiments. At the same time, the structures of the three Ndi1-substratecomplexes revealed the substrate binding modes in Ndi1, and demonstrated theexistence of two UQ binding sites. EPR (electron paramagnetic resonance) experimentsidentified for the first time the existence of semiquinone radicals, and the powersaturation experiments suggested that two semiquinone radicals are involved in electrontransport. Finally, a model was raised for the mechanism of electron transfer fromNADH to UQ catalyzed by Ndi1.
These results laid the foundation for the studies into the detailed mechanisms ofNDH-2-mediated electron transfer, NDH-2-targeted drug designs, and the application ofNdi1in gene therapy. This study incorporated research approaches from multiple disciplines including structural biology, biochemistry, molecular biology, cell biology,and physics. The research comprehensively revealed the mechanism how Ndi1functions as an entry point for electrons to traverse the respiratory chain.
本文以NDH-2家族的典型代表——酿酒酵母的Ndi1蛋白为研究对象，解析了其高分辨率晶体结构及Ndi1与NADH、UQ以及NADH-UQ三种底物复合物的晶体结构。在所有晶体结构中，Ndi1均以同源二聚体的形式存在。结构分析和序列比对发现，Ndi1具有一个独特的并且在NDH-2家族中序列高度保守的C端结构域（CTD），该结构域介导了Ndi1二聚体化，进而使其形成一个广泛的疏水区域，该区域赋予了Ndi1附着在线粒体内膜上的能力。后续的生物化学和细胞生物学实验都证实了CTD对于Ndi1功能的重要性。同时，三种Ndi1-底物复合物结构的解析揭示了Ndi1与底物的结合方式，并证明了Ndi1中同时存在两个泛醌结合位点。电子顺磁共振实验的结果首次鉴定出在Ndi1介导的电子传递过程中有半醌自由基的出现，同时功率饱和实验支持有两个半醌自由基参与电子传递反应的理论。据此，本论文提出了Ndi1介导电子传递的模型。
本文的研究成果为探讨NDH-2介导电子传递的详细机制、针对NDH-2的药物设计以及更好地将Ndi1应用于基因治疗奠定了良好的基础。本论文综合运用了结构生物学、生物化学与分子生物学、细胞生物学以及物理学等多个学科的实验方法系统地研究了NDH-2家族最具代表性的蛋白Ndi1的多方面特性，全方位地揭示了该蛋白发挥呼吸链电子传递入口这一重要生物学功能的机理。
The electron transport chain, or the respiratory chain, comprising of complexes I-V,transports the electrons derived from metabolic processes from NADH to the oxygenmolecule, and is the most important energy source for living organisms. Complex I, orNADH-ubiquinone oxidoreductase, is thus the entry point for these electrons. In manyorganisms, however, complex I could be replaced by type-II NADH dehydrogenases(NDH-2s), which catalyze the electron transfer from NADH to ubiquinone (UQ), withFAD as a prosthetic group. NDH-2s are highly conserved in many pathogenicmicroorganisms including Mycobacterium tuberculosis and Plasmodium falciparum,and have been recognized as potential drug targets. Meanwhile, heterologous expressionof Ndi1(an NDH-2in Saccharomyces cerevisiae) in human cells has been proposed totreat human diseases caused by complex I defects as typified by Parkinson’s diseases.
In this study, we have solved the high-resolution crystal structures of Ndi1and itsthree types of enzyme-substrate complexes with NADH, UQ and NADH-UQ,respectively. Ndi1formed a homodimer in all the structures. Structural analysis andsequence alignment identified that Ndi1possesses a C-terminal domain (CTD) which ishighly conserved among NDH-2s. The CTD mediates the homodimerization of Ndi1,thus generating an extensive hydrophobic region in the protein to facilitate itsmembrane attachment, which has been confirmed by the subsequent biochemical andcell biological experiments. At the same time, the structures of the three Ndi1-substratecomplexes revealed the substrate binding modes in Ndi1, and demonstrated theexistence of two UQ binding sites. EPR (electron paramagnetic resonance) experimentsidentified for the first time the existence of semiquinone radicals, and the powersaturation experiments suggested that two semiquinone radicals are involved in electrontransport. Finally, a model was raised for the mechanism of electron transfer fromNADH to UQ catalyzed by Ndi1.
These results laid the foundation for the studies into the detailed mechanisms ofNDH-2-mediated electron transfer, NDH-2-targeted drug designs, and the application ofNdi1in gene therapy. This study incorporated research approaches from multiple disciplines including structural biology, biochemistry, molecular biology, cell biology,and physics. The research comprehensively revealed the mechanism how Ndi1functions as an entry point for electrons to traverse the respiratory chain.
引文
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