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)是革兰氏杆菌外膜外叶的主要成分。 阴性菌如大肠埃希菌、鼠伤寒沙门氏菌等许多重要病原菌。LP (或内毒素)是在这一大类细菌中生存所必需的，是抵御 在宿主感染期间遇到的恶劣环境。鉴于内毒素在葛兰素史克生存中的重要作用- 阴性细菌--即，如果没有发生内毒素运输的任何步骤，细菌细胞就会死亡--而独特的细胞 它所创造的表面，详细了解参与内毒素合成和 运输将是开发针对这些有希望的新药靶点的新型抗生素的基础。 内膜(IM)ABC转运蛋白LptB2FG、LptB2FG、 膜锚定的周质蛋白LptC，周质蛋白LptA，推测形成一种 LptC和LptD之间的桥接以保护脂多糖在转运过程中的疏水酰链 周质，以及OM蛋白复合体LptDE，它将内毒素插入OM的外部小叶。在一个激动人心的时刻 目前，参与脂多糖转运的LPT系统中所有七种蛋白质的结构都已被解决。 值得注意的是，LPT系统的五个周质结构域显示出显著的结构同源性和晶体 结构为重要的内毒素转运过程的机制提供了有价值的见解，但它仍然 尚不清楚内毒素是如何通过革兰氏阴性细菌的周质LPT桥运输的。太棒了 已取得进展，包括确定脂多糖运输中的关键角色，确定晶体结构 对于每个LPT蛋白，建立桥模型，并鉴定和定量脂多糖结合部位 关于LPTA和LPTC，但关于这种关键的 革兰氏阴性细菌生理学中的分子。假设展开/展开的事件发生在 周质LPT蛋白使内毒素沿周质桥移动，并去除氨基酸侧链 对蛋白质-蛋白质和蛋白质-脂相互作用的稳定至关重要的是会扰乱体内的内毒素转运 通过互补的生物物理技术、计算研究和活体实验的组合进行测试 化验。成功完成拟议的目标将包括确定和量化 革兰氏阴性菌中内毒素转运的周质桥联组件的相互作用界面 脂多糖与LPT系统中每个周质结构域结合的机制和定量 对细菌中基本的内毒素运输过程的洞察。这项研究的长期目标是了解 参与脂多糖转运的蛋白质-蛋白质和蛋白质-配体相互作用使有效设计 选择性抑制内毒素在革兰氏阴性杆菌中转运的新药。