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。线粒体和叶绿体也存在类似的OMP生物发生机制，为这些细胞器与细菌的进化关系提供了进一步的证据。2012年，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(淋球菌和杜氏嗜血杆菌)的核心膜成分BAMA的晶体结构，BAMA本身是一种β-桶膜蛋白。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链的β-桶结构域。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β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可能会破坏沿着末端链的局部膜的稳定性，使膜变薄多达16埃。此外，这些MD模拟还揭示了BAMA的桶状结构域可以经历一个侧面的开口，从而创建一个从周质直接进入外膜的门户。这项研究发表在2013年的《自然》杂志上，随后的实验证实，BAM的功能需要横向打开Beta桶，这一结果发表在2014年的《结构》杂志上。目前的实验研究了4种BAM脂蛋白的作用以及它们是如何组装在一起并发挥作用的。为此，我们于2016年在《科学》杂志上发表了BAM复合体的结构。我们还在研究BAMA作为开发新抗生素的药物靶点的潜力，因为它是所有革兰氏阴性细菌的基本蛋白质。 随着BAM复合体所有组分结构的成功确定，我们现在将重点放在线粒体同系物、分选和组装机械、SAM复合体上。虽然Sam50和Bama被预测为结构和功能上的同源物，但SAM复合体的外围组件与BamB、C、D和E完全无关。SAM复合体组件的结构和功能表征将有助于阐明线粒体是如何进化到将蛋白质插入线粒体外膜的。在过去的一年里，我们在Sam50同源物的表达和纯化方面取得了重大进展；结晶实验正在进行中。 另一种处理线粒体蛋白的蛋白质复合体(包括运往SAM复合体的外膜蛋白)是外膜转位酶TOM复合体。我们正在研究外膜组件Tom40的结构和功能特征，最近与NICHD的Tatiana Rostovtseva和Sergey Bezrukov合作，通过电生理学完成了Tom40通道的功能特征。《生物化学杂志》正在审阅一份手稿。

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 mitochondria and chloroplasts, providing further evidence of the evolutionary relationship of these organelles to bacteria. In 2012, 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 remained elusive. To understand the mechanism of the BAM complex, we have determined crystal structures of the core membrane component called BamA, a beta-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 beta-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 beta 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 Angstroms. In addition, these MD simulations also revealed that the barrel domain of BamA can undergo a lateral opening to create a portal from the periplasm directly into the outer membrane. This work was published in Nature in 2013, with follow-up experiments confirming that lateral opening of the beta barrel is required for BAM function published in Structure, 2014. Current experiments investigate the roles of the 4 BAM lipoproteins and how they assemble and function together. Toward this end, we published the structure of the BAM complex in Science in 2016. We are also investigating the potential of BamA to serve as a drug target for the development of novel antibiotics, since it is an essential protein in all Gram-negative bacteria. With the successful structure determination of all components of the BAM complex, we are now focusing on the mitochondrial homolog, the Sorting and Assembly Machinery, SAM complex. While Sam50 and BamA are predicted to be structural and functional homologs, the peripheral components of the SAM complex are completely unrelated to BamB, C, D, and E. Structural and functional characterization of the SAM complex components will shed light on how mitochondria have evolved to insert proteins into the mitochondrial outer membrane. During the past year we have made significant progress in expression and purification of Sam50 homologs; crystallization experiments are in progress. Another protein complex that handles mitochondrial proteins (including the outer membrane proteins destined for the SAM complex), is the Translocase of the Outer Membrane, TOM complex. We are working on structural and functional characterization of the outer membrane component, Tom40, and have recently finished functional characterization of Tom40 channels by electrophysiology in collaboration with Tatiana Rostovtseva and Sergey Bezrukov at NICHD. A manuscript is under review at the Journal of Biological Chemistry.