A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA
A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA
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DOI：
10.1093/emboj/18.4.822
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发表时间：
1999-02-15
期刊：
影响因子：
11.4
通讯作者：
Bibi, E
中科院分区：
文献类型：
--
作者：
Edgar, R;Bibi, E
广泛的底物特异性现象的性质，表现为多药耐药蛋白，尚未了解。在大肠杆菌多药转运蛋白MdfA中，疏水性和PhoA融合分析到目前为止仅确定了一个膜包埋的带电荷氨基酸残基（E26），为了确定这个带负电荷的残基是否可能在多药识别中发挥作用，我们评估了在这个位置突变的MdfA构建体的表达和功能。用带正电荷的赖氨酸取代E26，可使氯霉素的耐药活性消失，但仍保留氯霉素的外排和耐药。相反，用天冬氨酸取代E26，可使氯霉素的识别和转运受到明显抑制;这些结果表明，尽管26位的负电荷对于主动转运不是必需的，它决定了MdfA的多药耐药特性，我们发现这种负电荷也存在于其他耐药转运蛋白中，并讨论了它在多药耐药方面的可能意义。
The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26), In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance, In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations, These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA, We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.