The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric
The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric
复制标题
DOI：
10.1074/jbc.m707899200
复制
发表时间：
2008-02-08
影响因子：
4.8
通讯作者：
Glaubitz, Clemens
中科院分区：
文献类型：
--
作者：
Lehner, Ines;Basting, Daniel;Glaubitz, Clemens
表现出广泛的底物特异性的转运蛋白是多药耐药现象的主要决定因素。大肠杆菌多药转运蛋白EmrE是一种4跨膜、螺旋状的12 kDa膜蛋白，形成功能性二聚体，以质子/药物反向转运的方式转运多种芳香族带正电荷的底物。在这里，我们报告的C-13化学位移的结合口袋内的基本残基Glu（14）。为了确保天然环境，EmrE被重组到E.大肠杆菌脂质。使用一维和二维双量子过滤的C-13固态NMR进行实验。为了明确分配Glu（14），引入E25 A突变以产生单个谷氨酸突变体。使用无细胞表达对Glu（14）进行C-13标记。通过质谱法、NMR光谱法、冷冻断裂电子显微镜法和转运测定法探测纯度、标记、均一性和功能性。对于Glu（14），观察到两组不同的化学位移，表明同二聚体EmrE结合口袋中的结构不对称性。在加入溴化乙锭后，观察到化学位移变化和改变的线形，证明二聚体中的两个Glu（14）与底物配位。
Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report C-13 chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered C-13 solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was C-13-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.