Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.
Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.
复制标题
LPS 的结合和转运是通过整个大肠杆菌 LPS 转运蛋白 LptA 上一系列位点的协调组合来实现的。
DOI：
10.1002/pro.4724
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Klug,CandiceS
中科院分区：
文献类型：
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作者：
Schultz,KathrynM;Schneider,JohnR;Fischer,MatthewA;Cina,NicholasP;Riegert,MollyO;Frank,DaraW;Klug,CandiceS
The outer leaflet of the outer membrane (OM) of bacteria such asEscherichia coli,Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram‐negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function ofE. coliLptA in vivo, we performed a systematic, unbiased, high‐throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of thelptAgene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss‐of‐function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within β‐strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.
