Institute of Biochemistry and Cluster of Excellence Frankfurt-Macromolecular Complexes, Goethe-University Frankfurt, D-60438 Frankfurt am Main, Germany.
Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5687-92. doi: 10.1073/pnas.1114944109. Epub 2012 Mar 26.
AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.
AcrAB-TolC 是大肠杆菌中主要的外排蛋白复合物，可将多种抗菌剂从细胞中排出。内膜成分 AcrB 是一个同源三聚体，据推测，单体以协同的方式连续循环经过三个构象阶段，分别命名为松散（L）、紧密（T）和开放（O）。已经在 T 构象的周质深结合口袋中显示出药物的结合。迄今为止，药物结合的初始步骤和向该药物结合位点的转运仍然难以捉摸。在这里，我们报告了与结合的米诺环素或阿霉素的 AcrB/设计的锚蛋白重复蛋白 (DARPin) 复合物的高分辨率结构（1.9-2.25 Å）。在 AcrB/阿霉素共晶结构中，明显结合了三个阿霉素分子，一个阿霉素分子结合在 T 单体的深结合口袋中，两个阿霉素分子以堆叠三明治的方式排列在 L 单体的侧周质裂缝中的进入口袋中。这个进入口袋与 T 单体中明显的深结合口袋由一个开关环隔开。这个环的定位和构象灵活性对于大底物似乎很重要，因为在大环内酯类转运中缺乏 G616N AcrB 变体表现出该环区域内的改变构象。转运似乎是一个逐步的过程，首先是在 L 单体的进入口袋中摄取药物，然后在 L 到 T 的转变过程中，药物在深结合口袋中得到容纳，进入 T 单体的内部深结合口袋。
