Substrate path in the AcrB multidrug efflux pump of Escherichia coli.
Substrate path in the AcrB multidrug efflux pump of Escherichia coli.
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DOI：
10.1111/j.1365-2958.2010.07330.x
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发表时间：
2010-10
影响因子：
3.6
通讯作者：
Nikaido H
中科院分区：
文献类型：
--
作者：
Husain F;Nikaido H
大肠杆菌的主要三方多药外排泵AcrAB-TolC赋予对多种化合物的抗性。药物分子可能从周质或周质/内膜界面被AcrB捕获，并通过AcrB再通过TolC到达介质。目前已有大量关于AcrB及其同源物可能与底物相互作用区域的晶体学和突变数据。从这些数据开始，我们设计了全细胞荧光实验，以确定通过AcrB的整个底物路径。我们沿着预测的底物路径测试了AcrB中的48个残基，其中25个给出了阳性结果，这是基于亲脂性染料马来酰亚胺对半胱氨酸残基的共价标记和用大容量马来酰亚胺试剂对尼罗河红外排的共价标记。这些残基都位于周围质域，我们将其指定为大的外部间隙的下部，间隙本身，晶体学定义的结合袋以及口袋和漏斗之间的门。我们的观察结果表明，底物被捕获在AcrB的较低间隙区域，然后通过结合袋，门，最终到达连接AcrB和TolC的AcrB漏斗。
A major tripartite multidrug efflux pump of Escherichia coli, AcrAB-TolC, confers resistance to a wide variety of compounds. The drug molecule is captured by AcrB probably from the periplasm or the periplasm/inner membrane interface, and is passed through AcrB and then TolC to the medium. Currently there exist numerous crystallographic and mutation data concerning the regions of AcrB and its homologs that may interact with substrates. Starting with these data, we devised fluorescence assays in whole cells to determine the entire substrate path through AcrB. We tested 48 residues in AcrB along the predicted substrate path and 25 gave positive results, based on the covalent labeling of cysteine residues by a lipophilic dye-maleimide and the blocking of Nile Red efflux by covalent labeling with bulky maleimide reagents. These residues are all located in the periplasmic domain, in regions we designate as the lower part of the large external cleft, the cleft itself, the crystallographically defined binding pocket, and the gate between the pocket and the funnel. Our observations suggest that the substrate is captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate, and finally to the AcrB funnel that connects AcrB to TolC.
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影响因子：
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作者：
Bohnert, Juergen A.;Schuster, Sabine;Kern, Winfried V.
通讯作者：
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影响因子：
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