Khan Ali G, Shouldice Stephen R, Tari Leslie W, Schryvers Anthony B
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada T2N 4N1.
Biochem J. 2007 Apr 1;403(1):43-8. doi: 10.1042/BJ20061589.
The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.
革兰氏阴性细菌病原体从转铁蛋白获取铁依赖于一种周质铁离子结合蛋白FbpA。FbpA将铁从外膜转运至内膜转运复合物。一个结合的磷酸根阴离子完成FbpA的铁配位壳层，动力学研究表明该阴离子在体外铁结合和释放中起关键作用。本研究旨在直接验证协同阴离子是完整细胞中铁转运所必需的这一假说。制备了一系列流感嗜血杆菌FbpA阴离子结合氨基酸位点的定点突变体（Gln-58、Asn-175和Asn-193），以提供在磷酸根阴离子结合方面有缺陷的蛋白质。各种突变体的晶体结构显示，C端结构域配体（Asn-175或Asn-193）而非N端结构域配体（Gln-58）的改变消除了磷酸根阴离子的结合。将突变蛋白导入流感嗜血杆菌以评估其介导铁转运的能力。所有单一位点定向突变体（Q58L、N175L和N193L）都能够介导从转铁蛋白和有限浓度的柠檬酸铁中获取铁。结果表明，FbpA介导的铁转运不依赖于协同阴离子结合位点中磷酸根的结合。
