Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019.
J Biol Chem. 2020 Apr 10;295(15):4974-4984. doi: 10.1074/jbc.RA119.011850. Epub 2020 Feb 25.
The outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N-C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N-C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.
外膜受体 FepA 通过能量和 TonB 依赖性但其他机制尚不清楚的过程运输三价 enterobactin (FeEnt)，该过程涉及其 150 个残基的内部 N 端球形结构域 (N 结构域)。我们在 FepA 三级结构的不同区域遗传引入了一对 Cys 残基，这些残基有可能形成二硫键。这些包括 N 结构域相邻 β-链上的 Cys 对（内 N）和将 N 结构域的外表面桥接到 C 端跨膜 β-桶内部的 Cys 对（内 N-C）。我们通过 siderophore 营养测试、[Fe]Ent 结合和摄取实验以及荧光诱饵传感器测定来表征这些突变体的 FeEnt 摄取情况。这三种方法一致表明，限制 N 结构域内构象运动的内 N 二硫键阻止了 FeEnt 的摄取，而大多数内 N-C 二硫键并没有阻止 FeEnt 的摄取。这些结果表明，在 FeEnt 转运过程中，FepA 的 N 末端必须发生构象重排。它们也反对 N 结构域作为刚体从通道中脱离，并表明它在 FeEnt 进入周质空间时仍留在跨膜孔内。
