铜绿假单胞菌LptD-LptE蛋白复合体的结构与功能研究

In Gram-negative bacteria, the outer leaflet of the outer membrane (OM) is composed predominantly of lipopolysaccharides (LPS) which are essential for the bacteria survival. The LptD-LptE complex plays a key role in OM biogenesis, by translocating newly synthesized LPS molecules from the periplasm into the external leaflet of the asymmetric OM during cell growth. The LptD in Pseudomonas aeruginosa (Pa) is a target for macrocyclic β-hairpin-shaped peptidomimetic antibiotics such as L27-11, which inhibits the transport of LPS to the cell surface. .To uncover the transport mechanism of LPS in P. aeruginosa and the mode of interaction between peptidomimetic compounds and its target LptD, we propose to determine the structures of the full length PaLptD-LptE and PaLptD-LptE-L27-11 complex to explore the mechanisms of LptD-LptE and to characterize the interaction of L27-11 with PaLptD-LptE using combinational methodologies which include X-ray crystallography, bacterial genetics and other biochemical and biophysical approaches. These studies may shed light on the transport and insertion mechanism of LPS, and open the door to new antibiotic strategies targeting the bacterial outer membrane.

革兰氏阴性细菌外膜的外小页主要由脂多糖分子组成。这一屏障可以有效地阻止有毒物质（如抗生素等）进入细菌，为绝大多数革兰氏阴性细菌的存活所必需。脂多糖在胞内合成以后，最后一步由LptD-LptE复合体负责把脂多糖从周质空间转运并装配到外膜的外小页。一类新研发的环状多肽药物（如L27-11），可通过靶向结合于LptD抑制细菌外膜的生成，从而有效地、特异性地杀死耐药的铜绿假单胞菌。我们拟利用X-射线衍射晶体学、细菌遗传学以及其他生物物理、生物化学等手段，对铜绿假单胞菌的LptD-LptE复合体从原子水平进行深入的结构学研究，探究LptD-LptE复合体的脂多糖转运机制与特点；并拟以铜绿假单胞菌LptD-LptE与环肽抗生素L27-11的共晶结构为基础，阐明二者的互作与抑菌机制，铜绿假单胞菌LptD-LptE蛋白复合体结构和功能的研究将为研发抗菌药物提供重要的靶点信息。

革兰氏阴性细菌外膜的外小页主要由脂多糖分子组成。脂多糖在胞内合成以后，由LptD-LptE复合体负责把脂多糖从周质空间转运并装配到外膜的外小页。新发现的环状多肽药物（L27-11），可特异靶向结合于LptD抑制细菌外膜的生成，从而有效地、特异性地杀死耐药的铜绿假单胞菌。我们利用X-射线衍射晶体学、细菌遗传学以及其他生物物理、生物化学等手段，对铜绿假单胞菌的LptD-LptE复合体展开相关结构学研究。我们成功解析出了PaLptD-LptE 全长3.28埃的晶体结构；我们发现paLptDE会特异结合脂蛋白，并成功解析出了paLptDE和脂蛋白YifL形成的复合体paLptDE/YifL 3.0 Å的晶体结构。我们对铜绿假单胞菌LptD-LptE蛋白复合体结构和功能的研究为研发抗菌药物提供重要的靶点信息。