The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3–4.1 Å and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA–OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development.

革兰氏阴性菌的高度不对称外膜在防御细胞毒性物质（如抗生素）中发挥作用。Mla 通路通过在内膜和外膜之间运输磷脂来维持外膜脂质的不对称性。它包含六种 Mla 蛋白 MlaFEDBCA，包括 ABC 转运蛋白 MlaFEDB，它通过未知机制发挥作用。在这里，我们确定了大肠杆菌的冷冻电镜结构MlaFEDB 处于 apo 状态并与磷脂、ADP 或 AMP-PNP 结合，分辨率为 3.3-4.1 Å，并建立了一个基于蛋白脂质体的转运系统，包括 MlaFEDB、MlaC 和 MlaA-OmpF，以监测磷脂的转运方向。体外转运测定和体内膜通透性测定结合诱变鉴定功能残基，这些残基不仅识别和转运磷脂，而且调节 MlaFEDB 复合物的活性和结构稳定性。我们的结果提供了对 Mla 途径的机制见解，这可能有助于抗菌药物的开发。