Conformational rearrangements in the N-domain of Escherichia coli FepA during ferric enterobactin transport.
Conformational rearrangements in the N-domain of Escherichia coli FepA during ferric enterobactin transport.
复制标题
铁肠杆菌素转运过程中大肠杆菌 FepA N 结构域的构象重排。
DOI：
10.1074/jbc.ra119.011850
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Klebba,PhillipE
中科院分区：
文献类型：
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作者：
Majumdar,Aritri;Trinh,Vy;Moore,KyleJ;Smallwood,ChuckR;Kumar,Ashish;Yang,Taihao;Scott,DanielC;Long,NoahJ;Newton,SaleteM;Klebba,PhillipE
大肠杆菌外膜受体FepA通过能量和TonB依赖性转运铁肠杆菌素（FeEnt），但其转运机制尚不明确，涉及其内部150个残基的N-末端球状结构域（N-domain）。我们在FepA三级结构的不同区域遗传地引入了成对的Cys残基，具有形成二硫键的潜力。这些包括N结构域的相邻β链上的Cys对（N内）和将N结构域的外表面桥接至C末端跨膜β桶内部的Cys对（N-C间）。我们的特点FeEnt摄取这些突变体铁载体营养试验，[59 Fe]Ent结合和摄取实验，和荧光诱饵传感器测定。这三种方法一致地表明，限制N结构域内构象运动的N内二硫键阻止FeEnt摄取，而大多数N-C间二硫键并不阻止FeEnt摄取。这些结果表明，构象重排必须发生在N末端的FepA在FeEnt运输。他们还反对N-结构域作为刚体脱离通道，并建议当FeEnt进入周质时，N-结构域保留在跨膜孔内。
TheEscherichia coliouter 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, [59Fe]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.