Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport
Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport
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DOI：
10.1074/jbc.m308893200
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发表时间：
2004-01-30
影响因子：
4.8
通讯作者：
Yamaguchi, A
中科院分区：
文献类型：
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作者：
Murakami, S;Tamura, N;Yamaguchi, A
我们先前报道了大肠杆菌中主要多药物输出蛋白AcrB的晶体结构（Murakami，S.，中岛河，Yamashita，E.，和Yamaguchi，A.（2002）Nature 419，587-593）。AcrB三聚体的膜外头部含有由三个α-螺旋组成的中心孔。每个螺旋孔属于不同的单体。在这项研究中，我们构建了半胱氨酸扫描突变体的残基组成的孔螺旋。在21个突变体（D99 C至P119 C）中，5个突变体（D101 C、V105 C、N109 C、Q112 C和P116 C）显示出显著降低的耐药性和药物输出活性。这些残基位于孔螺旋的一侧，即孔壁上。这些观察结果有力地表明了该孔在药物转运过程中的重要作用。N-乙基马来酰亚胺结合实验揭示孔处于闭合状态，并且孔中间的渗透性屏障的厚度对应于2.5个α-螺旋圈。两个突变体（V105 C和Q112 C），这表明所有的孔突变体的活性损失最大，在正常条件下检测到的二硫键交联二聚体的形式，这表明孔的构象变化是必不可少的运输过程中。
We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002) Nature 419, 587-593). The extramembrane headpiece of the AcrB trimer contains a central pore composed of three alpha-helices. Each pore helix belongs to a different monomer. In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix. Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity. These residues are localized on one side of the pore helix, i.e. on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 alpha-helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.