structural characterization of bacterial secretion channels

细菌分泌通道的结构特征

基本信息

批准号：
9356084
负责人：
Susan Buchanan
金额：
$ 179.02万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9356084
关键词：
Adopted Antibiotics Bacteria Biochemistry Biogenesis C-terminal Cell Survival Chloroplasts Collaborations Complex Crystallization Cytoplasm Development Drug Targeting Electrophysiology (science)Gram-Negative Bacteria Hemophilus ducreyi Homologous Gene Journals Lateral Light Lipid Bilayers Lipoproteins Manuscripts Membrane Membrane Proteins Mitochondria Mitochondrial Proteins Molecular Chaperones Molecular Conformation N-terminal Names National Institute of Child Health and Human Development Nature Neisseria gonorrhoeae Organelles Outer Mitochondrial Membrane Peptidoglycan Peripheral Process Protein translocation Proteins Publishing Role Science Signal Transduction Sorting - Cell Movement Structure Surface System TOM translocase Work beta barrel follow-up insight interest membrane biogenesis novel periplasm protein complex research study simulation translocase

项目摘要

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.

革兰氏阴性细菌、线粒体和叶绿体含有内膜和外膜。外膜含有大量通常称为外膜蛋白（OMP）的β-桶蛋白，它们在货物运输和信号传导中发挥重要作用，并且对于膜生物发生也至关重要。在革兰氏阴性细菌中，已知 OMP 在细胞质中合成，然后通过 Sec 易位子穿过内膜转运到周质。一旦进入周质，分子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 被一种称为 β 桶组装机械 (BAM) 复合体的复合体识别，该复合体折叠并将新的 OMP 插入外膜中。 BAM 复合体究竟如何实现其功能仍然未知。然而，我们确实知道 BAM 复合物由名为 BamA（本身就是 OMP）和 BamB、BamC、BamD 和 BamE 的五个成分组成，它们都是辅助脂蛋白。研究表明 BamA 和 BamD 对于细胞活力和 OMP 生物发生绝对重要。线粒体和叶绿体也存在类似的 OMP 生物发生机制，这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，而其他小组则解决了BamC、BamD、BamE以及BamA的大部分周质结构域。这些结构共同提供了对 BAM 复合物如何识别新生 OMP 的深入了解。然而，即使这些结构已知，BAM 复合物如何识别、折叠并将新生 OMP 插入外膜的机制仍然难以捉摸。为了了解 BAM 复合物的机制，我们确定了称为 BamA 的核心膜成分的晶体结构，BamA 本身是一种 β 桶膜蛋白，来自两个不同的物种（淋病奈瑟菌和杜克雷嗜血杆菌）。 BamA 的结构包含一个大的 N 端周质结构域和一个 C 端 16 链 β 桶结构域。周质结构域被发现有两种不同的构象，代表打开和关闭状态，这可以作为允许底物进入内部桶腔的门控机制。有趣的是，关闭状态伴随着末端β链的显着不稳定，发现末端β链隐藏在桶状结构域内。 MD 模拟显示，BamA 可以破坏末端链局部膜的稳定性，使膜变薄多达 16 埃。此外，这些 MD 模拟还表明，BamA 的桶状结构域可以经历横向开口，以创建从周质直接进入外膜的门户。这项工作于 2013 年发表在《Nature》杂志上，后续实验证实了 β 桶的横向开口是 BAM 功能所必需的，发表于《Structure》，2014 年。当前的实验研究了 4 种 BAM 脂蛋白的作用以及它们如何组装和一起发挥作用。。为此，我们于 2016 年在《科学》杂志上发表了 BAM 复合物的结构。我们还在研究 BamA 作为开发新型抗生素的药物靶点的潜力，因为它是所有革兰氏阴性细菌中的必需蛋白质。随着 BAM 复合体所有成分的结构成功确定，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 预计是结构和功能同源物，但 SAM 复合物的外围组件与 BamB、C、D 和 E 完全无关。SAM 复合物组件的结构和功能表征将揭示线粒体如何进化将蛋白质插入线粒体外膜。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（包括用于 SAM 复合物的外膜蛋白）的蛋白复合物是外膜转位酶，TOM 复合物。我们正在研究外膜成分 Tom40 的结构和功能表征，最近与 NICHD 的 Tatiana Rostovtseva 和 Sergey Bezrukov 合作，通过电生理学完成了 Tom40 通道的功能表征。《生物化学杂志》正在审查一份手稿。