Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA.
Department of Microbiology and Immunology, National University of Singapore, Singapore.
Mol Microbiol. 2018 Sep;109(5):633-641. doi: 10.1111/mmi.14002. Epub 2018 Sep 15.
Bacteria produce a variety of surface-exposed polysaccharides important for cell integrity, biofilm formation and evasion of the host immune response. Synthesis of these polymers often involves the assembly of monomer oligosaccharide units on the lipid carrier undecaprenyl-phosphate at the inner face of the cytoplasmic membrane. For many polymers, including cell wall peptidoglycan, the lipid-linked precursors must be transported across the membrane by flippases to facilitate polymerization at the membrane surface. Flippase activity for this class of polysaccharides is most often attributed to MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family proteins. Little is known about how this ubiquitous class of transporters identifies and translocates its cognate precursor over the many different types of lipid-linked oligosaccharides produced by a given bacterial cell. To investigate the specificity determinants of MOP proteins, we selected for variants of the WzxC flippase involved in Escherichia coli capsule (colanic acid) synthesis that can substitute for the essential MurJ MOP-family protein and promote transport of cell wall peptidoglycan precursors. Variants with substitutions predicted to destabilize the inward-open conformation of WzxC lost substrate specificity and supported both capsule and peptidoglycan synthesis. Our results thus suggest that specific substrate recognition by a MOP transporter normally destabilizes the inward-open state, promoting transition to the outward-open conformation and concomitant substrate translocation. Furthermore, the ability of WzxC variants to suppress MurJ inactivation provides strong support for the designation of MurJ as the flippase for peptidoglycan precursors, the identity of which has been controversial.
细菌产生多种表面暴露的多糖，这些多糖对于细胞完整性、生物膜形成和逃避宿主免疫反应至关重要。这些聚合物的合成通常涉及在细胞质膜内表面的脂质载体十一碳烯基磷酸上组装单体寡糖单位。对于许多聚合物，包括细胞壁肽聚糖，脂质连接的前体必须通过翻转酶跨膜运输，以促进在膜表面聚合。此类多糖的翻转酶活性通常归因于 MOP（多药/寡糖基-脂质/多糖）家族蛋白。对于这种普遍存在的转运蛋白如何识别和转运其同源前体跨越给定细菌细胞产生的许多不同类型的脂质连接寡糖，知之甚少。为了研究 MOP 蛋白的特异性决定因素，我们选择了参与大肠杆菌荚膜（甘露聚糖酸）合成的 WzxC 翻转酶的变体，该变体可以替代必需的 MurJ MOP 家族蛋白，并促进细胞壁肽聚糖前体的运输。预测会使 WzxC 内向开放构象不稳定的变体失去了底物特异性，并支持荚膜和肽聚糖的合成。因此，我们的结果表明，MOP 转运蛋白的特定底物识别通常会使内向开放状态不稳定，从而促进向外向开放构象的转变和伴随的底物转运。此外，WzxC 变体能够抑制 MurJ 失活，这为 MurJ 作为肽聚糖前体的翻转酶提供了强有力的支持，其身份一直存在争议。
