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首页> 外文期刊>Journal of bacteriology >Identification of Essential Charged Residues in Transmembrane Segments of the Multidrug Transporter MexB ofPseudomonas aeruginosa
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Identification of Essential Charged Residues in Transmembrane Segments of the Multidrug Transporter MexB ofPseudomonas aeruginosa

机译：铜绿假单胞菌多药转运蛋白MexB跨膜片段中基本带电残基的鉴定

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The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408→Glu/Lys939→Arg mutant retained significant activity, while an Asp407→Glu/Lys939→Arg mutant exhibited only marginal function. An Asp407→Glu/Asp408→Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407→Glu/Asp408→Glu/Lys939→Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.
机译：MexABM外排泵利用质子电化学梯度为铜绿假单胞菌赋予耐药性，从而输出结构多样的异生物素。 MexB亚基横穿内膜12次，在推定的跨膜片段2（TMS-2），TMS-4和TMS-10中分别具有两个，两个和一个带电残基。所有五个残基均发生突变，并通过测定抗生素的MIC和荧光染料外排来评估MexB功能。在TMS-2中，用Ala，Arg或Glu替代Lys342，用Ala，Gln或Asp替代Glu346并没有明显的效果。高度保守的TMS-4中Asp407的Ala，Asn或Lys取代导致活性丧失。此外，用Glu代替Asp407的突变体仅表现出边缘功能，这表明在该位置的侧链长度很重要。表现出显着功能的TMS-4中Asp408或TMS-10中Lys939的唯一替代物分别是Glu和Arg，这表明在这些位置需要天然电荷。另外，双中性突变体或带电荷残基Asp407和Lys939或Asp408和Lys939互换的突变完全丧失功能。 Asp408→Glu / Lys939→Arg突变体保留了显着的活性，而Asp407→Glu / Lys939→Arg突变体仅表现出边缘功能。 Asp407→Glu / Asp408→Glu双突变体也失去活性，但是通过用Arg替换Lys939（Asp407→Glu / Asp408→Glu / Lys939→Arg）而恢复了重要的功能。从总体上看，这些发现表明Asp407，Asp408和Lys939在功能上很重要，并增加了Asp407，Asp408和Lys939可能在TMS-4和TMS-10之间形成电荷网络的可能性，这对于质子转运和/或能量耦合。

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