Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany.
Hengyang Medical College, University of South China, Hengyang, Hunan Province, China.
Nat Commun. 2021 Jun 29;12(1):3889. doi: 10.1038/s41467-021-24151-3.
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
革兰氏阴性菌通过利用高效的外排泵来维持对有害物质进入的内在抵抗机制。大肠杆菌的 AcrAB-TolC 药物外排泵包含内膜 H/药物反向转运蛋白 AcrB，由三个功能上相互依赖的原体组成，在协同催化过程中连续循环通过松散（L）、紧密（T）和开放（O）状态。在这里，我们展示了运输循环中间状态的 13 个 AcrB 的 X 射线结构。基于结构的突变分析结合药物敏感性测定表明，药物是通过专门的运输通道引导到药物结合口袋。在红霉素、利奈唑胺、苯唑西林和夫西地酸的联合存在下获得的共结构显示夫西地酸在 T 原体内膜域深处结合。硫醇交联底物保护测定表明，该跨膜域结合位点也可以容纳苯唑西林或新生霉素，但不能容纳红霉素或利奈唑胺。在存在多种药物的情况下，AcrB 介导的药物转运被建议是别构调节的。
