Biogenesis of bacterial outer membrane proteins

为了深入了解OMP的生物发生，我们最近开发了一种方法来捕获大肠杆菌O157:H7自转运体的修饰形式，称为EspP，在组装后期稳定地结合到Bam复合体上。利用二硫键交联，我们发现当组装停止时，EspP β筒的c端β链与BamA （Bam复合物的中心亚基）β筒的横向“开放”形式的第一β链形成刚性界面。相比之下，EspP衍生物的n端β链与BamA最后两条β链的脂质面形成较弱的构象异质相互作用，可能代表中间组装状态。结果表明，BamA与客户蛋白在组装过程中形成了一个杂交桶状结构。基于我们的研究结果，我们提出了BamA通过一种新的“摆动”机制催化部分折叠的β桶的膜插入。

To obtain insight into OMP biogenesis, we recently developed a method to trap a modified form of an E. coli O157:H7 autotransporter called EspP stably bound to the Bam complex at a late stage of assembly. Using disulfide bond crosslinking, we found that when assembly stalls the C-terminal beta strand of the EspP beta barrel forms a rigid interface with the first beta strand of a laterally "open" form of the beta barrel of BamA, the central subunit of the Bam complex. In contrast, the N-terminal beta strand of the EspP derivative forms weaker, conformationally heterogeneous interactions with the lipid facing surface of the last two beta strands of BamA that likely represent intermediate assembly states. The results indicate that BamA forms a hybrid barrel with client proteins during their assembly. Based on our results, we proposed that BamA catalyzes the membrane insertion of partially folded beta barrels by a novel "swing" mechanism. We have also been using EspP as a model protein to study autotransporter biogenesis. In one major line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. It was originally proposed that the passenger domain is secreted through a channel formed by the covalently linked beta barrel domain (whence the name autotransporter), but results that we obtained from both biochemical and structural studies are inconsistent with this hypothesis. We found that the insertion of a small linker into the EspP passenger domain effectively creates a translocation intermediate by transiently stalling the translocation reaction (which is normally extremely rapid) near the site of the insertion. By using a site-specific photocrosslinking approach we found that residues adjacent to the stall point interact with BamA. These results support a model in which the Bam complex plays a major role in facilitating both the integration of the beta barrel domain into the OM and the translocation of the passenger domain across the OM. In a recent study, we found that the mutation of an unusual lipid-exposed lysine residue in the EspP beta barrel domain impairs a previously unidentified late folding step that follows both the membrane insertion of the beta barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that beta barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the beta barrel domain is fully assembled, our results also provide evidence against the hypothesis that autotransporters are autonomous protein secretion systems.