细菌通过特定的分泌系统将多种细胞内分子，包括药物、蛋白质和DNA，分泌到细胞外环境或宿主细胞。其中II型分泌系统广泛存在于革兰氏阴性菌中，它是唯一能够将蛋白底物以自然折叠状态分泌穿过细菌双层膜的分泌系统。II型分泌系统负责分泌多种病原菌的重要毒素，如霍乱毒素和肠毒素等，因此它在细菌致病机制中起着关键作用。II型分泌系统是一种多蛋白分子机器，横跨内外两层细胞膜。分泌素GspD蛋白在外膜上形成多聚体复合物通道，通过内膜复合物介导的能量转移，将假纤毛结构运送的底物分泌到细胞外环境。但是II型分泌系统的研究相对缺乏，所以一直难以了解这个复杂的分子机器是如何组装并分泌底物的。本文成功获得了全长的GspD复合物蛋白并分析了其近原子分辨率的结构，首次阐述了GspD的组装形式。同时本文还分析了GspD复合物突变体的结构，通过对结构的比较，再结合相应的生化和功能实验结果，合理地推测出底物分泌过程和通道开放机制。为了研究GspD蛋白在外膜上的定位机理，本文更进一步获得了AspS-GspD复合物蛋白并分析了其近原子分辨率的结构。首次看到了全长蛋白的复合物情况下，脂蛋白AspS与GspD蛋白以15：15的比例结合，结合位点包括S domain上的α11、loop和α12结构。通过引入突变的生化分析，本文发现639号苯丙氨酸位点对GspD与AspS蛋白的结合至关重要，未来可能作为潜在的药物靶点，通过影响GspD蛋白在外膜上的定位来抑制其毒素底物的分泌。同时本文还研究了内膜复合物，结果显示体外重组表达难以获得稳定的内膜复合物。虽然这部分暂时还没有取得可喜的研究成果，但是通过对研究结果的分析和失败原因的总结，未来可以更有针对性地进行实验设计，争取早日揭开内膜复合物的神秘面纱，最终能够完整全面地理解II型分泌系统的结构和功能。另外本文还进行了毒素复合物的研究，通过运用信号减去法对其复杂结构成功解析的经验可以应用到其他相似的结构解析案例中，同时也为实验室重构技术的开发提供了创新思路。毒素复合物的详细致病机理和作用通路还需进一步探索。对II型分泌系统外膜复合物的结构与功能研究填补了领域内的长期空白，为我们更全面地理解其底物的运输机制和设计特异性药物提供了重要的分子机理。

Bacteria secrete various intracellular molecules, including drugs, proteins and DNA, into the extracellular environment or host cells through specific secretion systems. Among them, type II secretion system (T2SS) widely existed in Gram-negative bacteria, which is the only one that can secrete protein substrate in the native folded state across the double membrane of Gram-negative bacteria. T2SS is responsible for secreting vital toxins of various pathogenic bacteria, such as cholera toxin and enterotoxin, and therefore plays a key role in bacterial pathogenesis. T2SS is a multiprotein nanomachine that spans the inner and outer membranes. Secretin GspD protein forms a polymeric complex channel on the outer membrane, and through the energy transfer mediated by inner membrane complex, the substrate transported by the pseudpilus is secreted by the GspD channel into the extracellular environment. However, the type II secretory system is relatively poorly studied, and it is difficult to understand how this nanomachine assembles and secretes substrate.In this paper, we successfully purified the complex of full-length GspD protein and analyze its structure at near-atomic resolution, and described the assembly form of GspD for the first time. Meanwhile, by analyzing the structures of the mutant of GspD complexes, and combining with the corresponding biochemical and functional experimental results, we reasonably speculated the gate-opening mechanism of the GspD channel in the process of secreting substrates.In order to study the localization mechanism of GspD protein on the outer membrane, we further obtained the protein of AspS-GspD complex and analyzed its structure at near atomic resolution. For the first time, in the case of full-length protein complex, lipoprotein AspS binds to GspD protein at a ratio of 15:15, and the binding sites of GspD protein contain α11, loop and α12 structures on S domain. By introducing mutations and their corresponding biochemical analysis, it was found that the phenylalanine 639 is crucial for GspD binding to AspS protein, and may be a potential drug target in the future to inhibit the secretion of toxin substrates by affecting the positioning of GspD protein on the outer membrane.At the same time, we also carried out experiments to study the inner membrane complex, and the results showed that it was difficult to obtain stable inner membrane complex by recombinant expression in vitro. Although this part temporarily haven't achieved gratifying results, the analysis of the results and possible reason for the failure may help us to design more suitable experiments and lift the veil of mysterious inner membrane complex. And finally we hope to give a comprehensive understanding for the structure and function of type II secretion system.In addition, this paper also studied toxin complex, and the experience of successfully resolving its complex structure by using the signal subtraction method can be applied to other similar structural analysis cases. At the same time, it also provides innovative ideas for the development of reconstruction technology in our laboratory. The detailed pathogenesis mechanism and target pathway of toxin complex are still need to be further explored.Studies on the structure and function of the outer-membrane complex in bacterial type II secretion system have filled in the long-term gaps in this field, providing important molecular mechanisms for a more comprehensive understanding of the substrates transport mechanism and the design of specific drugs.