structural characterization of bacterial secretion channels

革兰氏阴性细菌、线粒体和叶绿体含有内膜和外膜。外膜含有大量的β-桶蛋白，通常称为外膜蛋白(OMPS)，它们在货物运输和信号转导中起重要作用，对膜生物发生也是至关重要的。在革兰氏阴性细菌中，已知OMP是在细胞质中合成的，然后通过SEC易位子穿过内膜运输到周质中。一旦进入周质，伴侣就会引导新生的OMP穿过周质和肽聚糖到达外膜的内表面。在这里，新生的OMP由一种被称为Beta-Barrel组装机械(BAM)的复合体识别，该复合体折叠并将新的OMP插入外膜。BAM复合体到底是如何完成其功能的，目前尚不清楚。然而，我们知道BAM复合体由五个成分组成，分别为BAMA(OMP本身)和BAMB、BAMC、BAMD和BAME，它们都是辅助脂蛋白。研究表明，BAMA和BAMD对细胞存活和OMP生物发生是绝对必要的。线粒体和叶绿体都存在类似的OMP生物发生机制，进一步证明了这些细胞器的进化。最近，我们解决了BAMB的结构，而其他小组解决了BAMC，BAMD，BAME和BAMA的大部分周质结构域。这些结构共同为BAM复合体如何识别新生OMP提供了洞察力。然而，即使知道了这些结构，BAM复合体如何识别、折叠和将新生OMP插入外膜的机制仍然难以捉摸。 为了了解BAM复合体的机制，我们测定了来自两个不同物种(淋球菌和杜热氏嗜血杆菌)的核心膜成分BAMA的晶体结构，即桶状膜蛋白本身。BAMA的结构包含一个大的N-末端周质结构域和一个C-末端16链桶状结构域。周质结构域以两种不同的构象被发现，代表开放和关闭状态，这可能作为一种门控机制，允许底物进入内桶腔。有趣的是，关闭状态伴随着末端链的显著不稳定，它被发现隐藏在桶结构域中。MD模拟显示，BAMA可能会破坏末端链上的局部膜的稳定性，使膜变薄高达16%。此外，这些MD模拟还揭示了BAMA的桶状结构域可能经历了一个侧向开口，从而创建了一个从周质直接进入外膜的门户。这项工作正在《自然》杂志上发表。未来的实验将研究这4种BAM脂蛋白的作用，以及它们是如何组装和一起发挥作用的。

Gram-negative bacteria, mitochondria, and chloroplasts contain an inner and outer membrane. The outer membrane contains a host of beta-barrel proteins commonly called outer membrane proteins (OMPs), which serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. In Gram-negative bacteria, it is known that OMPs are synthesized in the cytoplasm and then transported across the inner membrane into the periplasm via a Sec translocon. Once in the periplasm, chaperones guide the nascent OMPs across the periplasm and peptidoglycan to the inner surface of the outer membrane. Here, the nascent OMPs are recognized by a complex known as the beta-barrel assembly machinery (BAM) complex which folds and inserts the new OMPs into the outer membrane. Exactly how the BAM complex is able to accomplish its function remains unknown. However, we do know that the BAM complex consists of five components named BamA (an OMP itself) and BamB, BamC, BamD, and BamE, which are all accessory lipoproteins. Studies have shown that BamA and BamD are absolutely essential for cell viability and OMP biogenesis. Similar mechanisms for OMP biogenesis exist for both mitochondria and chloroplasts, further evidence of the evolution of these organelles. Recently, we solved the structure of BamB, while other groups solved BamC, BamD, BamE and a large portion of the periplasmic domain of BamA. Together these structures provided insight into how the BAM complex may recognize nascent OMPs. However, even with these structures being known, the mechanism for how the BAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remains elusive. To understand the mechanism of the BAM complex, we have determined crystal structures of the core membrane component called BamA, a β-barrel membrane protein itself, from two different species (Neisseria gonorrhoeae and Haemophilus ducreyi). The structure of BamA contains a large N-terminal periplasmic domain and a C-terminal 16-stranded β-barrel domain. The periplasmic domain was found in two different conformations representing open and closed states, which may serve as a gating mechanism to allow substrate access to the internal barrel cavity. Interestingly, the closed state was accompanied by a significant destabilization of the terminal strand, which was found tucked inside the barrel domain. MD simulations revealed that BamA could destabilize the local membrane along the terminal strand, thinning the membrane by as much as 16 . In addition, these MD simulations also revealed that the barrel domain of BamA may undergo a lateral opening to create a portal from the periplasm directly into the outer membrane. This work is in press at Nature. Future experiments will investigate the roles of the 4 BAM lipoproteins and how they assemble and function together.