Biogenesis of bacterial outer membrane proteins

为了深入了解OMP的生物发生，我们开发了一种方法，在体内组装的后期阶段稳定地与Bam复合体结合，从而捕获一种名为ESPP的大肠杆菌O157：H7自转运蛋白的修饰形式。利用二硫键交联，我们发现当组装停止时，ESPPβ桶的C-末端β链(包含一个高度保守的基序称为β信号)与BAMA的β桶的第一个β链形成刚性界面，BAMA是Bam复合体的中央亚单位。相反，ESPP衍生物的N端Beta链与BAMA的最后两条Beta链形成较弱的构象异质相互作用，这两条链可能代表中间组装状态。最近，我们利用单粒子冷冻EM获得了与ESPP修饰形式结合的BAM络合物的高分辨率结构，并可视化了组装过程的动力学。与生化数据一致的是，低温电子显微镜数据表明，BAMAβ桶的第一条β链与ESPPβ信号形成了稳定的相互作用。结构数据还表明，ESPPβ桶的折叠是通过显著的杂化桶组装中间体进行的，在该中间体中，膜集成的β片层连接到BAMA上。这些结构显示出围绕ESPP组装中间体的膜发生了前所未有的偏转，表明弯曲的贝塔折叠逐渐向BAMA折叠，形成桶状结构。连同跟踪贝塔桶折叠的体内实验，当OM张力被修改时，我们的结果支持一个模型，在该模型中，Bam复合体利用OM弹性来加速贝塔桶折叠。 最近，我们还回顾了20多年前报道的一项令人惊讶的观察，即一种名为OmpA的八链大肠杆菌β桶蛋白在体内表达为两个非共价连接的片段时，可以组装成一个天然结构。在两个片段中引入单个半胱氨酸残基后，我们发现N-末端片段中的4个β链和C-末端片段中的5个β链之间的二硫键在周质空间中形成，极大地提高了分裂的OmpA的组装效率，但前提是半胱氨酸残基被完全注册。相反，我们只观察到N-末端片段中的β链1和C-末端片段中的8条β链之间的弱二硫键，这将形成闭合或环状排列的β桶。我们的结果不仅表明，贝塔桶在整合到OM之前就开始折叠成贝塔片状结构，而且有助于区分不同的OMP生物发生模式。 几年前，我们证明了仅使用含有纯化的BAM复合体和分子伴侣(SurA)的蛋白脂质体，就可以在体外重组小分子单体OMP的组装。最近，我们展示了我们可以通过重组更复杂的OMP的组装来扩展我们的检测的用途，三聚体孔蛋白OMPC。三聚体孔蛋白是大肠杆菌中含量最丰富的OMP之一，但人们对它们的组装知之甚少。我们发现，在体外合成的OMPC被插入到含有Bam复合体的蛋白脂质体中，并通过短暂的二聚体中间体折叠成热稳定的三聚体。有趣的是，OMPC的组装也依赖于脂多糖(LPS)的添加，这是一种专门位于OM中的糖脂。我们的结果强烈地表明，三聚体孔蛋白是通过一个逐步的过程形成的，这需要单体在组装能力的状态下整合到OM中。此外，我们的结果提供了令人惊讶的证据，表明与脂多糖的相互作用不仅是三聚反应所必需的，也是单体有效地插入到脂质双层中所必需的。

To obtain insight into OMP biogenesis, we 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 in vivo. Using disulfide bond crosslinking, we found that when assembly stalls the C-terminal beta strand of the EspP beta barrel (which contains a highly conserved motif known as the beta signal) 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 last two beta strands of BamA that likely represent intermediate assembly states. Recently, we used single-particle cryo-EM to obtain high-resolution structures of the Bam complex bound to the modified form of EspP and to visualize the dynamics of the assembly process. Consistent with the biochemical data, the cryo-EM data show that the first beta strand of the BamA beta barrel forms a stable interaction with the EspP beta signal. The structural data also show that the folding of the EspP beta barrel proceeds via remarkable hybrid-barrel assembly intermediates in which membrane integrated beta sheets are attached to BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP assembly intermediates and suggest that a curved beta sheet progressively folds towards BamA to form a barrel-like structure. Along with in vivo experiments that tracked beta barrel folding while the OM tension was modified, our results support a model in which the Bam complex harnesses OM elasticity to accelerate beta barrel folding. Recently, we also revisited a surprising observation reported over 20 years ago that an eight-stranded E. coli beta barrel protein called OmpA can be assembled into a native structure in vivo when it is expressed as two non-covalently linked fragments. After introducing single cysteine residues into the two fragments we found that disulfide bonds between beta strands 4 in the N-terminal fragment and 5 in the C-terminal fragment form in the periplasmic space and greatly increase the efficiency of assembly of split OmpA, but only if the cysteine residues are engineered in perfect register. In contrast, we observed only weak disulfide bonding between beta strands 1 in the N-terminal fragment and 8 in the C-terminal fragment that would form a closed or circularly permutated beta barrel. Our results not only demonstrate that beta barrels begin to fold into a beta sheet-like structure before they are integrated into the OM, but also help to discriminate between different models of OMP biogenesis. Several years ago we showed that the assembly of small, monomeric OMPs can be reconstituted in vitro using only proteoliposomes containing the purified Bam complex and a molecular chaperone (SurA). Recently, we showed that we could extend the utility of our assay by reconstituting the assembly of a more complex OMP, the trimeric porin OmpC. Trimeric porins are among the most abundant OMPs in E. coli, but their assembly is poorly understood. We found that in vitro synthesized OmpC was inserted into proteoliposomes that contained the Bam complex and folded into heat-stable trimers by passing through a short-lived dimeric intermediate. Interestingly, OmpC assembly was also dependent on the addition of lipopolysaccharide (LPS), a glycolipid located exclusively in the OM. Our results strongly suggest that trimeric porins form through a stepwise process that requires integration of monomers into the OM in an assembly-competent state. Furthermore, our results provide surprising evidence that an interaction with LPS is required not only for trimerization, but also for the productive insertion of monomers into the lipid bilayer.