Lpt protein-mediated transport of LPS

项目摘要 脂多糖（LPS）是革兰氏菌外膜（OM）外小叶的主要成分， 阴性细菌如大肠杆菌、鼠伤寒沙门氏菌和许多其他重要病原体。LPS (or内毒素）对于这一大类细菌的生存是必不可少的，并且作为抵抗细菌内毒素的第一道防线。 在宿主感染期间遇到的恶劣环境。考虑到LPS在革兰氏菌存活中的重要作用， 阴性细菌-即，如果LPS转运的任何步骤不发生，细菌细胞就会死亡， 它创建的表面，对LPS合成中涉及的蛋白质和机制的详细了解， 转运将是开发针对这些有前途的新药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LptB 2FG， 膜锚定的周质蛋白LptC，周质蛋白LptA，推测其形成一个 LptC和LptD之间的桥梁，以保护LPS的疏水性酰基链， 周质和OM蛋白复合物LptDE，其将LPS插入OM的外小叶中。在一个令人兴奋 随着研究的进展，参与LPS转运的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系统的五个周质结构域显示出显著的结构同源性， 结构提供了有价值的见解的机制，基本的LPS运输过程，但它仍然是 尚不清楚LPS是如何通过革兰氏阴性菌的假定周质Lpt桥转运的。伟大 研究取得了一些进展，包括确定LPS转运的关键参与者，确定LPS的晶体结构， 对于每种Lpt蛋白，建立桥模型，并鉴定和定量LPS结合位点 对LptA和LptC，但仍有许多问题，关于运输机制，这样一个关键的 革兰氏阴性细菌生理学中的分子。解折叠事件发生在 周质Lpt蛋白使LPS沿着周质桥移动， 对蛋白质-蛋白质和蛋白质-脂质相互作用的稳定至关重要的蛋白质-蛋白质和蛋白质-脂质相互作用将破坏LPS在体内的转运， 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。 测定。成功完成拟议目标将包括确定和量化 革兰氏阴性菌中脂多糖转运的周质桥组装体的相互作用界面和 LPS与Lpt系统中每个周质结构域结合的机制和定量， 深入了解细菌中的基本LPS运输过程。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， 选择性抑制革兰氏阴性病原体中LPS转运的新药。

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.