Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa
Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa
复制标题
DOI：
10.1128/jb.183.5.1734-1739.2001
复制
发表时间：
2001-03-01
影响因子：
3.2
通讯作者：
Nakae, T
中科院分区：
文献类型：
--
作者：
Guan, L;Nakae, T
MexABM外排泵输出结构多样的外源性物质，利用质子电化学梯度赋予铜绿假单胞菌耐药性。MexB亚基穿过内膜12次，并且在推定的跨膜区段2（TMS-2）、TMS-4和TMS- 10中分别具有两个、两个和一个带电残基。所有五个残基都被突变，并且通过确定抗生素的MIC和荧光染料流出来评价MexB功能。TMS-2中用Ala、Arg或Glu替换Lys 342和用Ala、Gln或Asp替换Glu 346没有可辨别的效果。TMS-4中的Asp 407被Ala、Asn或Lys取代，这是非常保守的，导致活性丧失。此外，用Glu代替Asp 407的突变体仅表现出边缘功能，表明该位置处的侧链长度是重要的。TMS-4中Asp 408或TMS-10中Lys 939表现出显著功能的唯一替代分别是Glu和Arg，表明这些位置处的天然电荷是必需的。此外，双中性突变体或其中带电残基Asp 407和Lys 939或Asp 408和Lys 939互换的突变体完全丧失功能。Asp 408->Glu/Lys 939->Arg突变体保留了显著的活性，而Asp 407->Glu/Lys 939->Arg突变体仅表现出边缘功能。Asp 407->Glu/Asp 4408->Glu双突变体也失去活性，但通过用Arg替换Lys 939（Asp 407->Glu/Asp 408->Glu/Lys 939->Arg）恢复了显著的功能。总的来说，这些发现表明Asp 407、Asp 408和Lys 939在功能上是重要的，并提高了Asp 407、Asp 408和Lys 939可能在TMS-4和TMS-10之间形成电荷网络的可能性，这对质子移位和/或能量偶联是重要的。
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, GIn, 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/ Asp4408->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.