Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.
Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.
Nat Commun. 2021 Aug 3;12(1):4687. doi: 10.1038/s41467-021-24965-1.
Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.
脂蛋白对于细菌的生长和抗生素耐药性至关重要。这些蛋白利用附着在 N 端半胱氨酸残基上的脂酰链锚定在细胞质膜的外表面。在革兰氏阴性菌中，许多脂蛋白被转运到外膜（OM），这一过程依赖于 ATP 结合盒（ABC）转运蛋白 LolCDE，该蛋白从细胞质膜中提取靶向 OM 的脂蛋白。脂锚定蛋白对转运机制构成了独特的挑战，因为它们既有疏水性脂质部分，又有可溶性蛋白成分，其潜在机制尚未完全了解。在这里，我们分别在无核苷酸和核苷酸结合状态下，以 3.8 Å 和 3.5 Å 的分辨率确定了嵌入纳米盘的 LolCDE 的冷冻电镜结构。结构分析，以及生化和突变研究，揭示了 LolCDE 如何通过与脂蛋白的脂质和 N 端肽部分相互作用来识别其底物，并确定酰胺连接的酰基链是 LolCDE 相互作用的关键元素。结合核苷酸后，LolCDE 的跨膜螺旋和周质域发生大规模、不对称运动，导致捕获的脂蛋白被挤出。LolCDE 和 MacB 的比较揭示了 VII 型 ABC 转运蛋白的保守机制，并强调了 LolCDE 作为三酰基化脂蛋白挤出器的独特性质。
