Yang Taihao, Zou Ye, Ng Ho Leung, Kumar Ashish, Newton Salete M, Klebba Phillip E
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, United States.
Front Microbiol. 2024 Mar 27;15:1355253. doi: 10.3389/fmicb.2024.1355253. eCollection 2024.
We studied the 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) (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 K 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 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 (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 FeEntin 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.
我们通过在其表面环中引入半胱氨酸残基并用荧光素马来酰亚胺（FM）对其进行修饰，研究了外膜蛋白Fiu，一种推测的单体儿茶酚铁转运蛋白。Fiu-FM结合了三儿茶酚铁载体肠杆菌素（FeEnt）和糖基化肠杆菌素（FeGEnt）的铁复合物、其二儿茶酚降解产物Fe（DHBS）（FeEnt*）、单儿茶酚二羟基苯甲酸（FeDHBA）和二羟基苯甲酰丝氨酸（FeDHBS），以及铁载体抗生素头孢地尔（FDC）和MB-1。与高亲和力配体门控孔蛋白（LGP）不同，Fiu-FM对铁复合物只有微摩尔亲和力。其对FeDHBS、FeDHBA、FeEnt*、FeEnt、FeGEnt、FeFDC和FeMB-1的表观K值分别为0.1、0.7、0.7、1.0、0.3、0.4和4μM。尽管Fiu具有广泛的结合能力，但其转运范围以及Cir和FepA的转运范围都较窄。Fiu只转运FeEnt*。Cir转运FeEnt和FeDHBS（较弱）；FepA转运FeEnt、FeEnt和FeDHBA。Cir和FepA都结合FeGEnt，尽管亲和力较低。相关转运蛋白（PiuA、PirA、BauA）对二价或单体儿茶酚铁也具有类似的中等亲和力和广泛特异性。微生物学和放射性同位素实验均表明Fiu只转运FeEnt*，而不转运单儿茶酚铁化合物。分子对接和分子动力学模拟预测了FeEnt在Fiu外部前庭的三个结合位点，以及其内部更深的第四个位点。最外层位点（1a、1b和2）的丙氨酸扫描诱变使FeEnt结合亲和力降低了20倍，并减少或消除了FeEnt的摄取。最后，分子动力学模拟提出了FeEnt通过Fiu的移动途径，这可能总体上描述了TonB依赖性受体的金属转运过程。
