Howard Hughes Medical Institute, University of Virginia School of Medicine, Charlottesville, VA, USA.
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
Nat Commun. 2022 Sep 5;13(1):5226. doi: 10.1038/s41467-022-32597-2.
O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.
O 抗原是革兰氏阴性外膜细胞外叶中脂多糖的普遍保护性延伸。在细胞质中合成后，脂质连接的多糖通过 WzmWzt ABC 转运体被运送到周质。通常，O 抗原的分泌需要其延伸末端的化学修饰，而转运体通过碳水化合物结合域（CBD）识别该末端。在这里，我们使用来自 A.aeolicus 的成分，确定了 O 抗原结构，其帽部分为甲基化甘露糖或鼠李糖。晶体和冷冻电子显微镜结构揭示了 WzmWzt 如何在无核苷酸状态下识别其 CBD 中的碳水化合物和核苷酸结合域之间的这个帽。ATP 结合诱导其 CBD 的剧烈构象变化，终止与 O 抗原的相互作用。ATP 酶测定和定点突变揭示了 O 抗原结合时水解活性降低，这可能有利于多糖加载到 ABC 转运体中。我们的结果阐明了 ABC 转运体识别和转运多糖的关键步骤。
