Yerushalmi H, Mordoch S S, Schuldiner S
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Biol Chem. 2001 Apr 20;276(16):12744-8. doi: 10.1074/jbc.M010979200. Epub 2001 Jan 19.
EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.
EmrE是一种来自大肠杆菌的多药转运蛋白，它通过与质子交换将有毒化合物从细胞中移除。Glu-14是假定膜结构域中唯一的带电荷残基，在该蛋白的50多个同源物中完全保守。该残基被证明是质子和底物共同的结合位点的重要组成部分。带有单个羧基残基Glu-14的EmrE表现出与野生型相似的摄取和结合特性。这表明一个仅含110个氨基酸且在第14位有单个羧基的小蛋白是表现出离子偶联转运活性的最基本结构。通过使用二环己基碳二亚胺（一种已知与羧基反应的疏水性碳二亚胺）来研究Glu-14在底物结合中的作用。二环己基碳二亚胺以剂量依赖性方式抑制四苯基鏻与野生型和单羧基突变体的结合。依他啶和EmrE的其他底物以与其表观亲和力一致的效力顺序阻止这种抑制。这表明底物在空间上阻止了二环己基碳二亚胺的结合，支持了反应性残基Glu-14是底物结合位点一部分的论点。
