Department of Molecular and Cellular Biology Biophysics Interdepartmental Group, University of Guelph, Guelph, ON N1G 2W1, Canada.
Mol Microbiol. 2012 Jun;84(6):1165-76. doi: 10.1111/j.1365-2958.2012.08084.x. Epub 2012 May 23.
Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophobic IM.
铜绿假单胞菌 PAO1 中的杂多聚 B 带 O-抗原（O-Ag）生物合成遵循 Wzy 依赖途径，从内膜（IM）的内叶到外叶开始转运十一异戊烯焦磷酸连接的阴离子 O-Ag 亚基（O 单位）。这种转运由整合的 IM 翻转酶 Wzx 介导。通过基于实验的无偏拓扑映射，我们小组之前观察到 Wzx 在其 12 个跨膜片段（TMS）内具有许多带电和芳香族氨基酸残基。在此，针对 Wzx 的 TMS 和环上的 102 个残基进行了定点突变，然后评估每个构建体恢复 B 带 O-Ag 产生的能力，确定了翻转酶功能的 8 个重要残基。突出了各种带电和芳香族残基的重要性，主要在蛋白质的 TMS 内，揭示了翻转酶内的功能“热点”，特别是在 TMS2 和 TMS8 内。Wzx 的三级结构同源模型的构建表明，TMS2 和 TMS8 构成中央阳离子腔。这是第一个描述带电荷的翻转酶腔介导阴离子 O 单位穿过疏水性 IM 转运的报告。
