Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking
Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking
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DOI：
10.1021/bi050452u
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发表时间：
2005-08-23
期刊：
影响因子：
2.9
通讯作者：
Yamaguchi, A
中科院分区：
文献类型：
--
作者：
Tamura, N;Murakami, S;Yamaguchi, A
acrabb - tolc系统输出多种药物和有毒化合物，并赋予大肠杆菌固有的药物耐受性。晶体结构表明AcrB和TolC直接对接。然而，直到最近，它们相互作用的生物化学和生物物理证据一直是矛盾的。在这项研究中，我们通过在AcrB的垂直发夹顶部和多组氨酸标记的TolC分子的卷曲线圈底部引入的位点定向诱变引入的半胱氨酸残基之间的体内二硫交联来检测相互作用位点，这些位点是结构上预测的对接位点。当一对特定的突变体在大肠杆菌中共表达时，检测到通过二硫交联形成的AcrB-TolC复合物。我们的观察结果表明，AcrB-TolC复合物可能通过AcrB和TolC的瞬时尖端相互作用两步机制形成。交联不受AcrA、底物或假定的质子偶联位点突变的影响。
The AcrAB-TolC system exports a wide variety of drugs and toxic compounds, and confers intrinsic drug tolerance on Escherichia coli. The crystal structures suggested that AcrB and TolC directly dock with each other. However, biochemical and biophysical evidence of their interaction has been contradictory until recently. In this study, we examine the interaction sites by means of in vivo disulfide cross-linking between cysteine residues introduced by site-directed mutagenesis at the tops of the vertical hairpins of AcrB and the bottoms of the coiled coils of polyhistidine-tagged TolC molecules, which are structurally predicted docking sites. The AcrB-TolC complex formed through disulfide cross-linking was detected when a specific pair of mutants was coexpressed in E. coli. Our observations suggested that the AcrB-TolC complex may be formed through a two-step mechanism via transient tip-to-tip interaction of AcrB and TolC. The cross-linking was not affected by AcrA, the substrate, or a putative proton coupling site mutation.