3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge
3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge
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DOI：
10.1021/bi051574p
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发表时间：
2005-11-15
期刊：
影响因子：
2.9
通讯作者：
Bibi, E
中科院分区：
文献类型：
--
作者：
Sigal, N;Vardy, E;Bibi, E
MdfA是大肠杆菌次级转运蛋白主要促进剂超家族（MFS）中的一种多药转运蛋白。虽然MdfA识别多药的几个方面已经被表征，但更好地理解其功能的详细机制需要结构信息。先前的研究基于LacY和G1pT的x射线结构，对MFS蛋白的3D结构进行了建模。然而，由于LacY、G1pT和MdfA之间的序列同源性差，需要额外的约束来建立可靠的同源性模型。使用预测每个跨膜螺旋（TM） （kPROT）的角取向的算法，我们获得了MdfA的12个TM与G1pT和LacY的非常相似的模式，表明它们都具有相似的螺旋填充。因此，使用kPROT结果作为附加约束，通过与LacY和G1pT进行结构比对，构建了MdfA的3D模型。通过相关突变分析和现有实验数据对模型进行了进一步的改进和初步评价。令人惊讶的是，除了先前表征的位于26位的膜嵌入谷氨酸外，该模型表明Asp34和Arg112位于膜内，与Glu26位于腔的同一面。重要的是，Arg112在二级药物转运体中是进化保守的，在这里我们发现这个位置的正电荷对于MdfA的多药转运是绝对必要的。
MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and G1pT. However, because of poor sequence homology, between LacY, G1pT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of G1pT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and G1pT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.