Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA–Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

膜蛋白外排泵通过挤出结构不同的化合物并降低其细胞内浓度来赋予抗生素抗性。然而，没有临床批准的药物来抑制外排泵，这将增强现有抗生素因药物外排而失效的功效。在这里，我们鉴定了抑制耐甲氧西林金黄色葡萄球菌的喹诺酮转运蛋白 NorA 的合成抗原结合片段 (Fab)(MRSA)。使用低温电子显微镜确定的两个 NorA-Fab 复合物的结构揭示了一个深深插入 NorA 底物结合口袋中的 Fab 环。该环上的精氨酸残基与两个相邻的天冬氨酸和谷氨酸残基相互作用，这些残基对 NorA 介导的 MRSA 抗生素耐药性至关重要。Fab 环的肽模拟物以亚微摩尔效力抑制 NorA，并与抗生素诺氟沙星联合消除 MRSA 生长。这些发现建立了一类肽抑制剂，可通过靶向 NorA 中不可或缺的残基来阻断 MRSA 中的抗生素流出，而无需膜通透性。