Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Department of Microbiology, The Ohio State University, Columbus, OH, USA.
Nature. 2019 Mar;567(7749):550-553. doi: 10.1038/s41586-019-1039-0. Epub 2019 Mar 20.
Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.
革兰氏阴性菌的细胞被内细胞质膜和外膜所包围，外膜起到保护屏障的作用，限制许多抗生素的进入。外膜的独特性质归因于脂多糖的存在。这种含有许多糖的大型糖脂在细胞质中合成；一组蛋白质形成膜到膜桥，介导脂多糖从内膜到细胞表面的运输。蛋白质桥的内膜成分包括一个 ATP 结合盒转运蛋白，为运输提供动力，但这个转运蛋白如何确保脂多糖单向穿过桥到外膜是未知的。在这里，我们描述了一个包含从膜中提取脂多糖并将其传递到蛋白质桥所需的所有蛋白质的五组件内膜复合物的两个晶体结构。对这些结构的分析，结合生化和遗传实验，确定了脂多糖进入转运蛋白腔并到达桥的路径。我们还确定了一个必须打开的蛋白质门，以允许底物从腔转移到桥上。脂多糖进入腔是不依赖于 ATP 的，但 ATP 是脂多糖通过门并转移到桥上所必需的。我们的发现解释了内膜转运复合物如何控制脂多糖的有效单向运输，克服其浓度梯度。
