structural characterization of bacterial secretion channels

细菌分泌通道的结构特征

• 批准号: 10248132
• 负责人: Susan Buchanan
• 金额: $ 136.16万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/10248132
• 关键词: Adopted; Affinity; Antibiotics; Bacteria; Binding; Biogenesis; Biological Assay; C-terminal; Cell Death; Cell Survival; Cells; Chloroplasts; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; Detergents; Development; Drug Targeting; Endoplasmic Reticulum; Gram-Negative Bacteria; Growth; Hemophilus ducreyi; Homologous Gene; Individual; Lateral; Light; Lipid Bilayers; Lipids; Lipoproteins; Manuscripts; Membrane; Membrane Proteins; Membrane Transport Proteins; Mitochondria; Mitochondrial Proteins; Molecular Chaperones; Molecular Conformation; Motion; Mutation; N-terminal; Names; Nature; Neisseria gonorrhoeae; Organelles; Outer Mitochondrial Membrane; Peptidoglycan; Peripheral; Process; Protein translocation; Proteins; Publishing; Research; Resolution; Role; Science; Signal Transduction; Sorting - Cell Movement; Structure; Surface; System; TOM translocase; Testing; Thinness; Toxin; Work; beta barrel; experimental study; follow-up; in vivo; insight; interest; membrane biogenesis; nanodisk; novel; particle; periplasm; protein complex; simulation; translocase

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. Ongoing research 2020: A recent extension of the BAM work is a new project to understand bacterial contact dependent growth inhibition, in which a bacterial two-partner secretion system binds to a BamA on a target bacterial cell for transfer of the toxin and eventual cell death. We have just solved the structures of two outer membrane transporters for the secretion partner(s) and are currently working to structurally and functionally characterize the entire system. We have developed an in vivo secretion assay to test various components and mutations and done MD simulations on the transporter. A manuscript describing this work is in revision at eLife. 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. We recently solved the first SAM complex structure at 3.2A resolution using cryo-EM, from single particles in detergent. We are completely this work by solving the same structure in lipid nanodiscs and anticipate submitting the manuscript within 3 months. Subsequent experiments will explore the functions of the individual subunits and the folding/insertion mechanism. 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 this complex, as well as the mitochondrial outer membrane protein Mdm10, which is involved in Tom40 biogenesis and lipid transfer from the endoplasmic reticulum. References: Noinaj, N., Fairman, J.W. & Buchanan, S.K. (2011). The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex. J. Mol. Biol., 407, 248-260. PMCID: PMC3048904 Noniaj, N., Kuszak, A.J., Gumbart, J.C., Lukacik, P., Chang, H., Easley, N.C., Lithgow, T. & Buchanan, S.K. (2013). Structural insight into the biogenesis of beta barrel membrane proteins. Nature 501: 385-390. PMCID:PMC3779476 Noinaj, N., Kuszak, A.J., Balusek, C. Gumbart, J.C. & Buchanan, S.K. (2014). Lateral opening and exit pore formation are required for BamA function. Structure 22:1055-62. PMCID: PMC4100585 Kuszak, A.J., Jacobs, D., Gurnev, P.A., Shiota, T., Louis, J., Lithgow, T., Bezrukov, S.M., Rostovtseva, T.K & Buchanan, S.K. (2015). Evidence of distinct channel conformations and substrate binding affinities for the mitochondrial outer membrane protein translocase pore Tom40. J. Biol. Chem. 290:26204-17. PMCID: PMC4646270 Bakelar, J., Buchanan, S.K. & Noinaj, N. (2016). The structure of the -barrel assembly machinery complex. Science 351:180-186. PMCID: PMC4883095 Noinaj N, Gumbart JC, Buchanan SK (2017) The -barrel assembly machinery in motion. Nat Rev Microbiol 15:197-204 PMCID: PMC5455337 Diederichs K.A., Ni X., Rollauer S.E., Botos I., Tan X., King M.S., Kunji E.R.S., Jiang J., Buchanan S.K. (2020). Structural insight into mitochondrial -barrel outer membrane protein biogenesis. Nat Commun. 2020 Jul 3;11(1):3290. doi: 10.1038/s41467-020-17144-1.PMID: 32620929 Free PMC article.

革兰氏阴性菌、线粒体和叶绿体都有内外膜。外膜含有大量通常称为外膜蛋白（OMPs）的β -桶蛋白，它们在货物运输和信号传导中起着重要作用，对膜生物发生也至关重要。在革兰氏阴性菌中，已知OMPs在细胞质中合成，然后通过Sec转座穿过内膜进入周质。一旦进入周质，伴侣蛋白引导新生的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）复合体识别，该复合体折叠并将新的omp插入外膜。确切地说，BAM复合体是如何完成其功能的仍然未知。然而，我们确实知道BAM复合体由五个组成部分组成，分别是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对细胞活力和OMP的生物发生至关重要。线粒体和叶绿体也存在类似的OMP生物发生机制，这进一步证明了这些细胞器与细菌的进化关系。2012年，我们解决了BamB的结构，而其他团队解决了BamC、BamD、BamE和BamA的大部分质周结构域。这些结构一起提供了BAM复合体如何识别新生omp的见解。然而，即使已知这些结构，BAM复合体如何识别、折叠并将新生的omp插入外膜的机制仍然难以捉摸。