Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.

药物流出是细菌和癌细胞中常见的耐药机制，但提供全面功能见解的研究很少。在这项研究中，我们对细菌 ABC 转运蛋白 EfrCD 进行了深度突变扫描 (DMS)，以确定 1,430 多个单一变体的药物流出活性概况。这些系统测量表明，在大底物结合口袋内的不同位置引入负电荷会导致向带正电荷的乙锭流出活性显着增加，而额外的芳香族残基则没有表现出相同的效果。在 EfrCD 的面向内的低温电子显微镜结构的背景下的数据分析发现了一个高亲和力结合位点，当转运蛋白转变为外向构象时，它通过蠕动转运机制释放结合的药物。最后，我们确定了导致 Hoechst 快速流入而不影响乙锭和柔红霉素流出活性的替代物。因此，单个突变可以将 EfrCD 转化为药物特异性 ABC 进口商。