Lpt protein-mediated transport of LPS

Lpt 蛋白介导的 LPS 转运

• 批准号: 10205081
• 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/10205081
• 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.

项目概要 脂多糖（LPS）是革兰氏菌外膜（OM）外层的主要成分。 阴性细菌，如大肠杆菌、鼠伤寒沙门氏菌和许多其他重要的病原体。 （或内毒素）对于这一大类细菌的生存至关重要，并且是抵抗细菌的第一道防线 鉴于LPS在革兰氏菌生存中的重要作用，宿主感染期间遇到的敌对环境。 阴性细菌 - 即如果 LPS 运输的任何步骤不发生，细菌细胞就会死亡 - 并且独特的细胞 它创造的表面，详细了解脂多糖合成和参与的蛋白质和机制 运输将成为开发针对这些有希望的新药物靶点的新型抗生素的基础。 七种蛋白质组成 LPS 转运 (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，推测其形成 LptC 和 LptD 之间的桥梁，以在运输过程中保护 LPS 的疏水酰基链 周质，以及将 LPS 插入 OM 外叶的 OM 蛋白复合物 LptDE。 随着LPS运输的进展，LPT系统中所有七种蛋白质的结构现已得到解决。 引人注目的是，LPt 系统的五个周质域表现出显着的结构同源性，并且晶体 结构为了解重要的 LPS 运输过程的机制提供了有价值的见解，但它仍然是 尚不清楚 LPS 如何穿过假定的革兰氏阴性细菌周质 Lpt 桥。 已取得进展，包括确定 LPS 运输的关键参与者、确定晶体结构 对于每种 Lpt 蛋白，开发桥模型，并识别和定量 LPS 结合位点 关于 LptA 和 LptC，然而关于这种关键物质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/折叠事件发生在 周质 Lpt 蛋白沿着周质桥移动 LPS 并去除氨基酸侧链 对蛋白质-蛋白质和蛋白质-脂质相互作用的稳定至关重要，会破坏体内 LPS 运输 通过互补的生物物理技术、计算研究和体内试验相结合进行测试 成功完成所提出的目标将包括鉴定和定量。 革兰氏阴性菌脂多糖转运周质桥组件的相互作用界面 LPS 与 Lpt 系统中每个周质结构域结合的机制和定量，以产生重要的结果 深入了解细菌中重要的脂多糖转运过程。这项研究的长期目标是了解。 LPS 转运中涉及的蛋白质-蛋白质和蛋白质-配体相互作用，以便能够有效设计 选择性抑制革兰氏阴性病原体中脂多糖转运的新药。