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

摘要 II型分泌系统（T2 SS）广泛存在于革兰氏阴性菌中，并输出 多种折叠蛋白质底物，例如来自霍乱弧菌的霍乱毒素（CT）和热- 产肠毒素大肠杆菌（ETEC）的不稳定肠毒素（LT）。T2 SS机械 它跨越整个细胞膜，由三层组成：神秘的内层 膜平台（IMP）、动态假纤毛和通道形成外膜 复杂.在目前的机制模型中，蛋白质底物与周质中的T2 SS结合， 触发细胞质ATP酶（GspE）水解ATP，从而促进 主要的假阿片肽亚基（GspG）转化为菌毛样结构。这个生长中的假纤毛 作为活塞或阿基米德螺旋，推动折叠的蛋白质底物通过外部 膜通道到细胞表面或细胞外环境。尽管经过几十年的研究， IMP的结构尚不可用;主要是由于这个原因，仍然不知道内部如何 膜组装平台将来自细胞质中ATP水解的能量转化为延伸 假纤毛并推动基质穿过外膜。也不知道是如何 内膜蛋白调节GspE ATP酶活性。我们的提议利用了 荧光尺寸排阻色谱和协同进化分析，以确定相互作用 我们将使用辅助多聚体策略和非天然氨基 酸交联以纯化稳定的内膜蛋白复合物，然后单颗粒 冷冻电子显微镜来确定复杂的结构。我们将分析ATP酶活性 GspE与不同的内膜蛋白复合物。该提案将大大 增加有关结构和机制的基本科学知识， T2 SS，从而为开发未来针对T2 SS的治疗干预措施提供新的基础 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.