结核病的主要致病菌是结核分枝杆菌，作为一种常见的慢性传染性疾病，在包括中国在内的许多发展中国家中，仍然是引发较高死亡人数的疾病之一。近年来，耐药结核的出现更对结核病的治疗带来了新的挑战，而呼吸链复合物II由于结构保守性较强及在呼吸链和三羧酸循环中的重要功能，成为了理想的药物设计靶点之一。解析高分辨率复合物II的三维结构，可以为抗结核药物的研发提供良好的结构理论基础。 本研究工作通过冷冻电镜技术解析了耻垢分枝杆菌（结核分枝杆菌的模式菌）呼吸链复合物II原子级别分辨率结构，总体分辨率为2.84?。复合物II的整体结构以同源三聚体的形式存在，在每个单体中，除较为保守的SdhA、SdhB、SdhC和SdhD亚基之外，一个全新的跨膜亚基SdhF也被鉴定出来，该亚基由一根跨膜螺旋构成，通过阻塞近端的醌结合位点发挥功能，并通过与磷脂分子的相互作用稳定了跨膜区的整体结构。质谱结果及相关的结构分析显示，心磷脂、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酸等脂质分子在复合物II整体结构的稳定中发挥了重要作用。与此同时，在每个单体中鉴定出了两个位于远端的醌结合位点，其中一个醌结合位点位于三体内部，由相邻单体的不同SdhD亚基所形成，参与的主要氨基酸包括Gln94、Met98、Trp102、Tyr146及Thr150等；另外一个位于单体外侧，以相对独立的口袋形式呈现，参与的主要氨基酸包括Phe60、Phe67、Trp72、Trp93及Trp96等。在琥珀酸：泛醌氧化还原酶活性实验中，通过比较野生型蛋白复合体同突变蛋白复合体的活性差异，这两个醌结合位点在电子传递过程中发挥的功能得到了初步验证。在以上研究基础上，我们提出了耻垢分枝杆菌复合物II三聚体协同电子传递的机制，解释了三聚体功能发挥的重要生理意义，对醌结合位点的结构分析也为以复合物II为靶点的抗结核药物设计奠定了基础。
Mycobacterium tuberculosis is the main pathogen of tuberculosis. As a common chronic infectious disease, tuberculosis is still the main cause of patients and deaths in many developing countries, including China. In recent years, the emergence of drug-resistant tuberculosis has brought new challenges to the tuberculosis treatment. Respiratory chain complex II has become one the ideal drug design targets due to its high structural conservation and important functions in respiratory chain and tricarboxylic acid cycle. Therefore, the determination of high resolution structure of complex II could provide solid theoretical basis for the development of anti-TB drugs. In this paper, the atomic resolution structure of complex II from Mycobacterium smegmatis (model strain of Mycobacterium tuberculosis) was determined by cryo-EM method, with an overall resolution 2.84 ?. The structure of complex II exists in a trimer form, while in each monomer, apart from the conservative subunits SdhA, SdhB, SdhCand SdhD, a new transmembrane subunit SdhF is also identified. SdhF subunit, composed of one transmembrane helix, functions by blocking the proximal quinone binding site and stabilizes the whole transmembrane domains by interacting with phospholipid molecules.The results of mass spectrometry and structural analysis shows that phosopholipids, including cardiolipin, phosphatidyl ethanolamine, phosphatidyl inositol and phosphatidylic acid, could play an important role in the stability of overall structure. At the same time, two distal quinone binding sites are identified in each monomer. One site is located inside the trisomy, formed by different SdhD subunits of adjacent monomers and the amino acids involved in this interaction include Gln94, Met98, Trp102, Tyr146 and Thr150. The other is located on the outer side of the monomer and presents as a relatively independent pocket, with the nearby amino acids Phe60, Phe67, Trp72, Trp93 and Trp96. In the experiments of succinate:ubiquinone oxidoreductase activity, thefunctions of these two quinone binding sites in electron transport are preliminarily verified by comparing the enzyme activity differences between wild-type proteins and mutant proteins. Based on the above research, we propose the mechanism of trimercooperative electron transfer in Mycobacterium smegmatis complex II, and explain theimportant physiological significance of trimer formation. The structural analysis of quinone binding sites also lays a foundation for the design of anti-tuberculosis drugs targeting complex II.
