Unraveling the mechanism by which the BAM complex mediates OMP biogenesis

项目概要 革兰氏阴性细菌，例如大肠杆菌，具有外膜，可提供营养并添加 对细胞的保护。外膜还含有蛋白质，可以使病原菌 逃避宿主免疫反应并引起许多严重的传染病。尽管 这些外膜蛋白（OMP）对于细胞存活和毒力的重要性，人们知之甚少 这些蛋白质如何折叠并插入外膜。然而，在大肠杆菌中，我们知道 200 kDa 五组分复合物，称为 β 桶组装机械 (BAM) 复合物，介导 这些膜蛋白的生物发生。在过去的十年中，各个组件的单独结构 据报道，BAM 复合物以 BamA 为中心成分，提供了最 关于 BAM 复合体如何发挥作用的有用线索。据透露，BamA 可能会破坏稳定 局部膜，从而促进蛋白质折叠和插入膜中。最近，我们的工作 等人确定了 BAM 复合物的结构，揭示了前所未有的构象 BamA 桶域内的变化。我们在这里的工作将利用这些结构来研究 BAM 复合物在 OMP 生物发生中的功能的构象变化。此外，我们将 确定底物 OMP 如何被 BAM 复合物识别以及它们如何与 BAM 相互作用 在折叠成膜的过程中。最后，我们将利用结构生物学方法来研究BAM复合体 在脂质双层中并且在底物存在下。我们提出的研究将提供关键的功能和 揭示 BAM 复合物如何在细菌中发挥其重要作用的结构见解。

Project Summary Gram-negative bacteria, such as E. coli, have an outer membrane which provides nutrients and added protection for the cell. The outer membrane also contains proteins that can enable pathogenic bacteria to evade host immune responses and cause a number of serious infectious diseases. Despite the importance of these outer membrane proteins (OMPs) for cell survival and virulence, little is known about how these proteins are folded and inserted into the outer membrane. However, in E. coli, we know that a 200 kDa five-component complex called the β-barrel assembly machinery (BAM) complex mediates the biogenesis of these membrane proteins. In the last decade, the individual structures of components of the BAM complex have been reported with the structure of BamA, the central component, providing the most useful clues to how the BAM complex may function. Here, it was revealed that BamA may destabilize the local membrane, thereby promoting protein folding and insertion into the membrane. Recently, our work and others’ have determined the structure of the BAM complex, revealing unprecedented conformational changes within the barrel domain of BamA. Our work here will utilize these structures to study the role of the conformational changes for the function of the BAM complex in OMP biogenesis. Additionally, we will determine how substrate OMPs are recognized by the BAM complex and how they interact with BAM during folding into the membrane. Lastly, we will use structural biology methods to study the BAM complex in a lipid bilayer and in the presence of substrates. Our proposed studies will provide crucial functional and structural insight towards unraveling how the BAM complex performs its essential role in bacteria.