PDF(Pubmed)
Abstract：
We studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt) and glucosylated enterobactin (FeGEnt), their dicatecholate degradation product Fe(DHBS)2 (FeEnt*), the monocatecholates dihydroxybenzoic acid (FeDHBA) and dihydroxybenzoyl serine (FeDHBS), and the siderophore antibiotics cefiderocol (FDC) and MB-1. Unlike high-affinity ligand-gated porins (LGPs), Fiu-FM had only micromolar affinity for iron complexes. Its apparent KD values for FeDHBS, FeDHBA, FeEnt*, FeEnt, FeGEnt, FeFDC, and FeMB-1 were 0.1, 0.7, 0.7, 1.0, 0.3, 0.4, and 4 μM, respectively. Despite its broad binding abilities, the transport repertoires of E. coli Fiu, as well as those of Cir and FepA, were less broad. Fiu only transported FeEnt*. Cir transported FeEnt* and FeDHBS (weakly); FepA transported FeEnt, FeEnt*, and FeDHBA. Both Cir and FepA bound FeGEnt, albeit with lower affinity. Related transporters of Acinetobacter baumannii (PiuA, PirA, BauA) had similarly moderate affinity and broad specificity for di- or monomeric ferric catecholates. Both microbiological and radioisotopic experiments showed Fiu\'s exclusive transport of FeEnt*, rather than ferric monocatecholate compounds. Molecular docking and molecular dynamics simulations predicted three binding sites for FeEnt*in the external vestibule of Fiu, and a fourth site deeper in its interior. Alanine scanning mutagenesis in the outermost sites (1a, 1b, and 2) decreased FeEnt* binding affinity as much as 20-fold and reduced or eliminated FeEnt* uptake. Finally, the molecular dynamics simulations suggested a pathway of FeEnt* movement through Fiu that may generally describe the process of metal transport by TonB-dependent receptors.
摘要：
我们研究了大肠杆菌外膜蛋白Fiu，一种假定的单体儿茶酚铁转运蛋白，通过在其表面环中引入Cys残基并用荧光素马来酰亚胺（FM）对其进行修饰。三甲酚酸铁载体肠杆菌素（FeEnt）和糖基化肠杆菌素（FeGEnt）的Fiu-FM结合的铁复合物，它们的二甲酸降解产物Fe(DHBS)2(FeEnt*)，单甲酸酯二羟基苯甲酸（FeDHBA）和二羟基苯甲酰基丝氨酸（FeDHBS），和铁载体抗生素头孢地洛（FDC）和MB-1。与高亲和力配体门控孔蛋白（LGP）不同，Fiu-FM对铁络合物仅具有微摩尔亲和力。FeDHBS的表观KD值，FeDHBA,FeEnt*,FeEnt,FeGEnt,FeFDC,和FeMB-1分别为0.1、0.7、0.7、1.0、0.3、0.4和4μM，分别。尽管它具有广泛的约束力，大肠杆菌Fiu的运输库，还有Cir和FepA,不那么广泛。Fiu仅运输FeEnt*。Cir运输了FeEnt*和FeDHBS（弱）；FepA运输了FeEnt，FeEnt*,和FeDHBA。Cir和FepA都绑定了FeGEnt,尽管亲和力较低。鲍曼不动杆菌的相关转运蛋白（PiuA，皮拉,BauA）对二-或单体儿茶酚铁具有类似的中等亲和力和广泛特异性。微生物和放射性同位素实验均显示了Fiu对FeEnt*的独家运输，而不是单茶酚铁化合物。分子对接和分子动力学模拟预测了Fiu外部前庭中FeEnt*的三个结合位点，和其内部更深的第四个地点。最外层位点的丙氨酸扫描诱变（1a，1b,和2)降低FeEnt*结合亲和力多达20倍,并降低或消除FeEnt*摄取。最后,分子动力学模拟提出了FeEnt*通过Fiu运动的途径，该途径通常可以描述TonB依赖性受体的金属运输过程。
