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一种修饰形式的E。大肠杆菌O 157：H7自转运蛋白EspP在组装后期稳定结合到Bam复合物上。使用二硫键交联，我们发现当组装停止时，EspP β桶的C-末端β链与BamA β桶的横向“开放”形式的第一β链形成刚性界面，BamA β桶是BAM复合物的中心亚基。相比之下，EspP衍生物的N-末端β链与BamA的最后两条β链的面向脂质的表面形成较弱的构象异质性相互作用，这可能代表中间组装状态。结果表明，BamA在组装过程中与客户蛋白形成了一个混合桶。基于我们的研究结果，我们提出BamA催化膜插入的部分折叠β桶的一种新的“摆动”机制。 我们也一直使用EspP作为模型蛋白来研究自转运蛋白的生物合成。在一个主要的调查路线，我们一直在研究的机制，其中EspP乘客域是跨OM易位。最初提出乘客结构域通过共价连接的β桶结构域形成的通道分泌（因此命名为自转运蛋白），但我们从生化和结构研究中获得的结果与这一假设不一致。我们发现，将小接头插入到EspP乘客结构域中通过在插入位点附近暂时停止易位反应（其通常非常快速）有效地产生易位中间体。通过使用特定位点的光交联方法，我们发现邻近失速点的残基与BamA相互作用。这些结果支持了一个模型，其中BAM复合物在促进β桶结构域整合到OM和乘客结构域跨OM的易位中起着重要作用。在最近的一项研究中，我们发现EspP β桶结构域中一个不寻常的脂质暴露赖氨酸残基的突变损害了一个先前未识别的晚期折叠步骤，该步骤在β桶结构域的膜插入和乘客结构域的分泌之后，但在蛋白水解成熟之前。我们的研究结果表明，β桶组装可以在插入后阶段完成，并提高了与膜脂质的相互作用可以促进体内折叠的可能性。此外，通过显示乘客结构域被分泌之前，β桶结构域完全组装，我们的研究结果也提供了证据，反对的假设，autotorpers是自主的蛋白质分泌系统。

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.