Lpt protein-mediated transport of LPS

Lpt 蛋白介导的 LPS 转运

• 批准号: 9816218
• 负责人: CANDICE S KLUG
• 金额: $ 35.42万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816218
• 关键词: ATP-Binding Cassette Transporters; Affect; Affinity; Amino Acids; Antibiotics; Bacteria; Bacterial Physiology; Binding; Binding Sites; Biological Assay; Biophysics; C-terminal; Calorimetry; Carrier Proteins; Cell surface; Cells; Cessation of life; Complex; Computational Technique; Crystallization; Data; Development; Dimerization; Disease; Drug Design; Electron Spin Resonance Spectroscopy; Endotoxins; Environment; Escherichia coli; Event; Excision; Foundations; Future; Goals; Gram-Negative Bacteria; Growth; Human; Hydrophobicity; Infection; Inflammatory; Lead; Ligands; Lipids; Lipopolysaccharides; Mediating; Membrane; Membrane Proteins; Microbiology; Modeling; Modification; Molecular Conformation; Mutation; N-terminal; Names; Periplasmic Proteins; Protein Analysis; Proteins; Pseudomonas aeruginosa; Research; Role; Salmonella typhimurium; Septic Shock; Side; Spectrum Analysis; Structure; Surface Plasmon Resonance; System; Techniques; Testing; Titrations; Transport Process; Work; biophysical analysis; biophysical techniques; computer studies; design; dimer; hydrophilicity; in vivo; innovation; insight; invention; light scattering; membrane biogenesis; mutant; new therapeutic target; novel; novel therapeutics; pathogen; pathogenic bacteria; periplasm; pressure; protein complex; protein protein interaction; protein transport; structural biology

Project Summary Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane (OM) of Gram- negative bacteria such as Escherichia coli, Salmonella typhimurium and many other important pathogens. LPS (or endotoxin) is essential for survival in this large class of bacteria and serves as a first line of defense against hostile environments encountered during host infection. Given the essential role of LPS in the survival of Gram- negative bacteria – i.e., the bacterial cells die if any step of LPS transport does not occur – and the unique cell surface it creates, a detailed understanding of the proteins and mechanisms involved in LPS synthesis and transport will be the foundation on which to develop novel antibiotics against these promising new drug targets. Seven proteins make up the LPS transport (Lpt) system: the inner membrane (IM) ABC transporter LptB2FG, the membrane-anchored periplasmic protein LptC, the periplasmic protein LptA, which is speculated to form a bridge between LptC and LptD to protect the hydrophobic acyl chains of LPS during transport through the periplasm, and the OM protein complex LptDE that inserts LPS into the outer leaflet of the OM. In an exciting advance, the structures of all seven proteins in the Lpt system involved in LPS transport have now been solved. Strikingly, the five periplasmic domains of the Lpt system show remarkable structural homology and the crystal structures provide valuable insights into the mechanism of the essential LPS transport process, yet it is still unknown how LPS is transported across the putative periplasmic Lpt bridge of Gram-negative bacteria. Great progress has been made, including identifying the key players in LPS transport, determining the crystal structures for each of the Lpt proteins, developing the bridge model, and identifying and quantitating the LPS binding site on LptA and LptC, and yet many questions remain regarding the mechanism of transport of such a critical molecule in Gram-negative bacterial physiology. The hypothesis that unfolding/folding events occur in the periplasmic Lpt proteins to move LPS along the periplasmic bridge and that removal of amino acid side chains critical to the stabilization of the protein-protein and protein-lipid interactions will disrupt LPS transport in vivo will be tested through a combination of complementary biophysical techniques, computational studies and in vivo assays. The successful completion of the proposed aims will include the identification and quantitation of the interaction interfaces of the periplasmic bridge assembly for LPS transport in Gram-negative bacteria and the mechanism and quantitation of LPS binding to each periplasmic domain in the Lpt system to yield important insights into the essential LPS transport process in bacteria. The long-term goal of this research is to understand the protein-protein and protein-ligand interactions involved in LPS transport to enable the effective design of novel drugs to selectively inhibit LPS transport in Gram-negative pathogens.

项目摘要 脂多糖（LPS）是革兰氏外膜（OM）外叶的主要组成部分 阴性细菌，例如大肠杆菌，鼠伤寒沙门氏菌和许多其他重要病原体。 LPS （或内毒素）对于在这种大类细菌中生存至关重要，并且是对抗的第一道防线 宿主感染期间遇到的敌对环境。鉴于LP在革兰事的生存中的基本作用 阴性细菌 - 即，如果没有发生LPS的任何步骤，细菌细胞会死亡 - 独特的细胞 它产生的表面，对LPS合成涉及的蛋白质和机制的详细理解和 运输将是针对这些有希望的新药物靶标开发新型抗生素的基础。 七个蛋白质组成了LPS Transper（LPT）系统：内膜（IM）ABC转运蛋白LPTB2FG， 膜锚定的周质蛋白LPTC，周质蛋白LPTA，据推测形成A LPTC和LPTD之间的桥梁，以保护LPS的疏水酰基链在运输过程中 Pariplasm和OM蛋白复合物LPTDE将LPS插入OM的外部小叶中。令人兴奋 进步，现在已经解决了参与LPS运输的LPT系统中所有七种蛋白质的结构。 引人注目的是，LPT系统的五个周围域显示出显着的结构同源性和晶体 结构为基本LPS运输过程的机制提供了宝贵的见解，但仍然是 未知的LP如何在革兰氏阴性细菌的假定周质LPT桥上运输。伟大的 已经取得了进展，包括确定LPS运输的主要参与者，确定晶体结构 对于每个LPT蛋白，开发桥模型，并识别和定量LPS结合位点 在LPTA和LPTC上，但是关于如此关键的运输机制仍然存在许多问题 革兰氏阴性细菌生理学中的分子。展开/折叠事件的假设发生在 周质LPT蛋白沿周质桥移动LP，并去除氨基酸侧链 对于稳定蛋白质蛋白质和蛋白质脂质相互作用至关重要 通过互补生物物理技术，计算研究和体内的结合进行测试 测定。拟议的目标的成功完成将包括对 革兰氏阴性细菌中LPS运输的外围桥组件的相互作用界面和 LPT系统中LPS结合LPS的机制和定量，以产生重要 深入了解细菌中必需的LPS运输过程。这项研究的长期目标是了解 LPS运输中涉及的蛋白质 - 蛋白质蛋白和蛋白质 - 配体相互作用，以实现有效的设计 在革兰氏阴性病原体中有选择地抑制LPS运输的新型药物。