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来捕获一种修饰形式的E。大肠杆菌自转运蛋白EspP在体内组装后期稳定结合到Bam复合物上。在大肠在大肠杆菌中，Bam复合物由BamA和四种脂蛋白（BamB-BamE）组成，BamA是在所有革兰氏阴性细菌中发现的蛋白质，其由16链跨膜β桶结构域和五个周质多肽转运相关（POTRA）结构域组成。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（其含有被称为β信号的高度保守基序）与BamA β桶的侧向开放形式的第一β链形成刚性界面。相比之下，EspP衍生物的N-末端β链与BamA的最后两条β链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。最近，我们使用单粒子cryo-EM获得高分辨率结构的BAM复杂绑定到修改后的形式的EspP和可视化的动态组装过程。与生物化学数据一致，cryo-EM数据显示BamA β桶的第一条β链与EspP β信号形成稳定的相互作用。结构数据还表明，EspP β桶的折叠通过显著的“混合桶”组装中间体进行，其中膜整合的β片层连接到BamA。这些结构显示出围绕EspP组装中间体的膜的前所未有的偏转，并表明弯曲的β片层逐渐向BamA折叠以形成桶状结构。沿着体内实验，跟踪β桶折叠，同时修改OM的张力，我们的结果支持一个模型，其中BAM复合体利用OM的自然刚性来加速β桶折叠。 最近发现了几种抗菌化合物，它们可以有效地抑制必需BamA蛋白的活性，但它们的作用方式尚不清楚。为了解决这个问题，我们研究了三种抑制剂（一种称为darobactin的天然产物和两种称为Polyphor peptide 7和MRL-494的合成化合物）对体内EspP生物合成的影响。我们发现darobactin阻断EspP β信号与BamA的结合，但如果在结合后阶段加入，则对组装没有影响。相比之下，Polyphor肽7和MRL-494抑制β信号与BamA的结合以及至少一个后续组装步骤。结合先前分析darobactin和Polyphor peptide 7与BamA体外相互作用的研究，我们的结果强烈表明这两种化合物通过不同的竞争和变构机制抑制BamA功能。除了提供对抗菌化合物性质的深入了解外，我们的结果还提供了直接的实验证据，支持β信号与BamA的结合启动OMP膜插入的模型。 BamA是在革兰氏阴性细菌、线粒体和叶绿体中发现的OMP的Omp 85超家族的成员，其通过存在独特的16链β桶结构域和至少一个POTRA结构域来定义。迄今为止已经研究的所有Omp 85蛋白都促进关键的OMP组装和/或蛋白质易位反应。铜绿假单胞菌PlpD是一个很大程度上未表征的Omp 85蛋白家族的原型，该蛋白家族含有N-末端马铃薯糖蛋白样（PL）结构域以及POTRA结构域和C-末端β桶结构域。部分基于PlpD家族与另一Omp 85蛋白家族的明显相似性，人们普遍认为PL结构域通过共价连接的β桶结构域易位到细胞外空间（在那里它作为毒力因子发挥作用）。与目前的教条相反，我们发现PlpD PL结构域仅存在于周质中，并且与先前研究的单体Omp 85蛋白不同，PlpD形成同源二聚体。值得注意的是，PL结构域包含一个片段，该片段通过与相邻的β桶结构域进行瞬时链交换而表现出前所未有的动态性。我们的研究结果表明，Omp 85超家族的结构比目前认为的更多样化，并表明Omp 85支架在进化过程中被利用来产生新的功能。

To obtain insight into OMP biogenesis, we developed a method to trap a modified form of an E. coli autotransporter called EspP stably bound to the Bam complex at a late stage of assembly in vivo. In E. coli, the Bam complex consists of BamA, a protein found in all Gram-negative bacteria that consists of a 16-stranded transmembrane beta barrel domain and five periplasmic polypeptide transport associated (POTRA) domains, and four lipoproteins (BamB-BamE). 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 BamA beta barrel. 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 tension of the OM was modified, our results support a model in which the Bam complex harnesses the natural rigidity of the OM to accelerate beta barrel folding. Several antibacterial compounds have been discovered recently that potently inhibit the activity of the essential BamA protein, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors (a natural product called darobactin and two synthetic compounds called Polyphor peptide 7 and MRL-494) on the biogenesis of EspP in vivo. We found that darobactin blocked the binding of the EspP beta signal to BamA, but had no effect on assembly if added at a post-binding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both the binding of the beta signal to BamA and at least one later step of assembly. Taken together with previous studies that analyzed the interaction of darobactin and Polyphor peptide 7 with BamA in vitro, our results strongly suggest that the two compounds inhibit BamA function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the beta signal to BamA initiates the membrane insertion of OMPs. BamA is a member of the Omp85 superfamily of OMPs found in Gram-negative bacteria, mitochondria and chloroplasts that are defined by the presence of a distinctive 16-stranded beta barrel domain and at least one POTRA domain. All of the Omp85 proteins that have been studied to date promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of a largely uncharacterized Omp85 protein family that contains an N-terminal patatin-like (PL) domain as well as a POTRA domain and a C-terminal beta barrel domain. Based in part on the apparent similarity of the PlpD family to another Omp85 protein family, it is widely believed that the PL domain is translocated into the extracellular space (where it functions as a virulence factor) by the covalently linked beta barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and that unlike previously studied Omp85 proteins which are monomeric, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the adjacent beta barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.