Unraveling the mechanism by which the BAM complex mediates OMP biogenesis

揭示 BAM 复合物介导 OMP 生物发生的机制

• 批准号: 10415950
• 负责人: Nicholas Noinaj
• 金额: $ 29.63万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-08 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415950
• 关键词: Affinity; Antibiotics; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; CSPG6 gene; Cell Membrane Proteins; Cell Survival; Cell physiology; Communicable Diseases; Complex; Cryoelectron Microscopy; Crystallization; Cytoprotection; Detergents; Development; Escherichia coli; Genetic; Gram-Negative Bacteria; Immune response; In Vitro; Individual; Infection; Integral Membrane Protein; Lateral; Libraries; Lipid Bilayers; Lipoproteins; Mediating; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Molecular Machines; Multi-Drug Resistance; Multiprotein Complexes; Mutate; Nutrient; Pathogenicity; Play; Process; Protein Truncation; Proteins; Reporting; Resolution; Roentgen Rays; Role; Signal Transduction; Structure; Virulence; Virulence Factors; Work; base; biophysical analysis; crosslink; extracellular; in vivo; insight; interest; membrane biogenesis; nanodisk; pathogenic bacteria; protein folding; protein function; recruit; resistant strain; structural biology

Project Summary Gram-negative bacteria, such as E. coli, have an outer membrane which provides nutrients and added protection for the cell. The outer membrane also contains proteins that can enable pathogenic bacteria to evade host immune responses and cause a number of serious infectious diseases. Despite the importance of these outer membrane proteins (OMPs) for cell survival and virulence, little is known about how these proteins are folded and inserted into the outer membrane. However, in E. coli, we know that a 200 kDa five-component complex called the β-barrel assembly machinery (BAM) complex mediates the biogenesis of these membrane proteins. In the last decade, the individual structures of components of the BAM complex have been reported with the structure of BamA, the central component, providing the most useful clues to how the BAM complex may function. Here, it was revealed that BamA may destabilize the local membrane, thereby promoting protein folding and insertion into the membrane. Recently, our work and others’ have determined the structure of the BAM complex, revealing unprecedented conformational changes within the barrel domain of BamA. Our work here will utilize these structures to study the role of the conformational changes for the function of the BAM complex in OMP biogenesis. Additionally, we will determine how substrate OMPs are recognized by the BAM complex and how they interact with BAM during folding into the membrane. Lastly, we will use structural biology methods to study the BAM complex in a lipid bilayer and in the presence of substrates. Our proposed studies will provide crucial functional and structural insight towards unraveling how the BAM complex performs its essential role in bacteria.

项目摘要 革兰氏阴性细菌，如大肠杆菌，有一层外膜，提供营养并增加 对细胞的保护。外膜还含有蛋白质，可以使病原菌 逃避宿主免疫反应，引发多种严重传染病。尽管 这些外膜蛋白(OMP)对细胞存活和毒力的重要性，人们知之甚少 这些蛋白质是如何折叠并插入外膜的。然而，在大肠杆菌中，我们知道一种 200 kDa的五组分复合体称为β-Barrel组装机械复合体，调节着 这些膜蛋白的生物发生。在过去的十年中，各组成部分的个别结构 据报道，BAM复合体的中心成分BAMA的结构提供了最多 关于BAM复合体如何发挥作用的有用线索。在这里，据透露，巴马可能会破坏 局部膜，从而促进蛋白质折叠和插入膜。最近，我们的工作 和其他人已经确定了BAM复合体的结构，揭示了前所未有的构象 在巴马的桶域内发生了变化。我们的工作将利用这些结构来研究 BAM复合体在OMP生物发生中功能的构象变化。此外，我们还将 确定底物OMP如何被BAM复合体识别以及它们如何与BAM相互作用 在折叠到膜中的过程中。最后，我们将使用结构生物学方法来研究BAM复合体 在脂质双层中和在底物存在的情况下。我们提议的研究将提供关键的功能和 对BAM复合体如何在细菌中发挥其基本作用的结构性洞察。