Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

中文翻译：

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MsbA介导的脂多糖转运的结构基础

革兰氏阴性细菌外膜中的脂多糖（LPS）对于其细胞包膜的组装至关重要。内膜细胞质小叶中合成的LPS通过ATP结合盒转运蛋白MsbA翻转到周质小叶中。尽管付出了巨大的努力，但MsbA驱动的LPS翻转背后的结构机制仍然难以捉摸。在这里，我们使用单粒子冷冻电子显微镜阐明在三种功能状态下脂质纳米糖包埋的MsbA的结构。不含核苷酸的MsbA跨膜结构域的4.2分辨率结构显示，LPS在周质小叶的高度深深地结合在MsbA内部，与MsbA建立了广泛的亲水和疏水相互作用。具有ADP钒酸盐和ADP的MsbA的两个亚纳米分辨率结构分别显示出前所未有的封闭构象和向内构象。我们的研究揭示了LPS识别的结构基础，描绘了MsbA向翻转LPS的构象转变，并为其他脂质翻转酶的结构表征铺平了道路。