Chaptal Vincent, Zampieri Veronica, Wiseman Benjamin, Orelle Cédric, Martin Juliette, Nguyen Kim-Anh, Gobet Alexia, Di Cesare Margot, Magnard Sandrine, Javed Waqas, Eid Jad, Kilburg Arnaud, Peuchmaur Marine, Marcoux Julien, Monticelli Luca, Hogbom Martin, Schoehn Guy, Jault Jean-Michel, Boumendjel Ahcène, Falson Pierre
Drug Resistance and Membrane Proteins Group, Molecular Microbiology and Structural Biochemistry Laboratory, CNRS UMR 5086, University of Lyon, IBCP, 7, passage du Vercors, 69367 Lyon, France.
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
Sci Adv. 2022 Jan 28;8(4):eabg9215. doi: 10.1126/sciadv.abg9215. Epub 2022 Jan 26.
Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg-bound outward-facing conformations of the (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in . Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.
多药ABC转运蛋白通过一种药物释放的分子特征迄今未知的机制将药物转运穿过细胞膜。在这里，我们通过X射线晶体学和单颗粒冷冻电子显微镜（EM）在去污剂溶液中解析了（同二聚体）BmrA的三种ATP-Mg结合外向构象，其中一种与罗丹明6G（R6G）结合，R6G是BmrA在[具体生物]中过表达时输出的一种底物。两个R6G分子在外小叶水平结合到药物结合腔，位于一个单体的跨膜（TM）螺旋1-2和另一个单体的TM5'-6'之间。它们诱导TM1-2重排，突出了我们通过氢/氘交换和分子动力学模拟证实的局部灵活性。在没有R6G的情况下，模拟显示由疏水性驱动的腔快速释放后堵塞，而当存在R6G时，R6G可以在腔内移动，保持腔开放。
