Structural analysis of inner membrane platform in the type 2 secretion system

摘要 第二型分泌系统(T2SS)广泛存在于革兰氏阴性菌中，并输出 各种折叠蛋白底物，如霍乱弧菌霍乱毒素(CT)和热- 产肠毒素大肠杆菌(ETEC)的不稳定肠毒素(LT)。T2SS机器 横跨整个细胞外壳，由三个部件组成：神秘的内部 膜平台(IMP)、动态假性纤毛和通道形成外膜 很复杂。在目前的机制模型中，蛋白质底物与周质中的T2SS结合 触发细胞质ATPase(GspE)水解三磷酸腺苷，从而使 将主要的伪匹林亚基(GSPG)转变为菌毛状结构。这种不断增长的假性纤毛表现为 就像活塞或阿基米德螺丝一样，推动折叠的蛋白质底物穿过外部 通向细胞表面或细胞外环境的膜通道。尽管进行了数十年的研究，但 IMP的结构还没有得到，主要是因为这个原因，还不知道内部是如何 膜组装平台将细胞质内ATP水解所产生的能量转化为延伸 假乳头并将底物推过外膜。也不知道它是如何 内膜蛋白调节GspE-ATPase活性。我们的建议充分利用了 荧光尺寸排除层析和协同进化分析用于鉴定相互作用 在IMP的成分中。我们将使用辅助多聚体策略和非天然氨基 酸交联法纯化稳定的内膜蛋白复合体 低温电子显微镜来确定复杂的结构。我们将分析ATPase的活性 不同内膜蛋白复合体对GspE的影响。这项提议将极大地 增加有关体系结构和机制的基本科学知识 T2SS，从而为开发未来针对A2SS的治疗干预提供了新的基础 广泛的细菌感染。

ABSTRACT The type II secretion system (T2SS) exists widely in gram-negative bacteria and exports a variety of folded protein substrates, such as cholera toxin (CT) from Vibrio cholerae and heat- labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC). The T2SS machinery spans the entire cell envelope and consists of three subassemblies: the mysterious inner membrane platform (IMP), the dynamic pseudopilus, and the channel-forming outer membrane complex. In current mechanism models, protein substrate binding to the T2SS in the periplasm triggers the cytoplasmic ATPase (GspE) to hydrolyze ATP, which energizes the incorporation of the major pseudopilin subunit (GspG) into a pilus-like structure. This growing pseudopilus acts as a piston or Archimedes screw, pushing the folded protein substrates through the outer membrane channel to the cell surface or extracellular milieu. Despite decades of research, the structure of IMP is not yet available; mainly for this reason, it is still unknown how the inner membrane assembly platform converts energy from ATP hydrolysis in the cytoplasm to extend the pseudopilus and push the substrates across the outer membrane. It is also unknown how the inner membrane proteins regulate the GspE ATPase activity. Our proposal exploits fluorescence size exclusion chromatography and coevolution analysis to identify interactions among components of IMP. We will use an assistant-multimer strategy and unnatural amino acid crosslinking to purify stable inner membrane protein complexes, followed by single particle cryo-electron microscopy to determine complex structures. We will analysis the ATPase activity of GspE with different inner membrane protein complexes. This proposal will substantially increase the fundamental scientific knowledge about the architecture and mechanisms of the T2SS, and thereby provide a new basis for developing future therapeutic interventions against a broad range of bacterial infections.