Lipopolysaccharide binding to the periplasmic protein LptA
Lipopolysaccharide binding to the periplasmic protein LptA
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DOI：
10.1002/pro.3177
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发表时间：
2017-08-01
期刊：
影响因子：
8
通讯作者：
Klug, Candice S.
中科院分区：
文献类型：
--
作者：
Schultz, Kathryn M.;Lundquist, Tanner J.;Klug, Candice S.
脂多糖（LPS）和质周蛋白（LptA）是革兰氏阴性菌的两个重要组成部分。脂多糖又称内毒素，不对称分布于革兰氏阴性菌如大肠杆菌的外膜外小叶中，参与机体在不利环境条件下的自然防御。LptA是脂多糖转运蛋白（Lpt）家族的一员，该家族还包括LptC、LptDE和LptBFG(2)，其功能是在MsbA翻转LPS穿过内膜后，将LPS通过周质转运到外膜的外小叶。假设LPS与LptA结合穿过外周质，LPS的酰基链与LptA的中心袋结合。本文所描述的研究是第一个全面表征和定量LPS与LptA结合的研究。利用位点定向自旋标记电子顺磁共振（EPR）技术，收集LptA上LPS结合袋内及周围15个自旋标记残基的数据，观察外源LPS存在引起的迁移率变化，确定LPS与LptA的结合位置。获得的EPR数据表明LPS:LptA复合物的比例为1:1，并允许首次计算LptA-LPS相互作用的解离常数。结果表明，整个蛋白受到LPS结合的影响，n端在LPS存在下展开，并且突变的LptA蛋白无法形成低聚物，其对LPS的亲和力发生了改变。
Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG(2), that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.