Characterization and Iron Binding Dynamics of Haemophilus Influenzae Ferric Binding Protein

The periplasmic FbpA (ferric-binding protein A) from Haemophilus influenzae plays a critical role in acquiring iron from host transferrin, shuttling iron from the outer-membrane receptor complex to the inner-membrane transport complex responsible for transporting iron into the cytoplasm. In the present study, we report on the properties of a series of site-directed mutants of two adjacent tyrosine residues involved in iron co-ordination, and demonstrate that, in contrast with mutation of equivalent residues in the N-lobe of human transferrin, the mutant FbpAs retain significant iron-binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron, but are proficient in mediating periplasm-to-cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized Escherichia coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor-bound transferrin. Wild-type iron-loaded FbpA could be crystalized in a closed or open state depending upon the crystallization conditions. The synergistic phosphate anion was not present in the iron-loaded open form, suggesting that initial anchoring of iron was mediated by the adjacent tyrosine residues and that alternate pathways for iron and anion binding and release may be considered. Collectively, these results demonstrate that the presence of a twin-tyrosine motif common to many periplasmic iron-binding proteins is critical for initially capturing the ferric ion released by the outer-membrane receptor complex.

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Characterization of a ferric‐binding protein mutant in Haemophilus influenzae

Molecular Microbiology ◽

10.1111/j.1365-2958.1997.mmi535.x ◽

1997 ◽

Vol 25 (5) ◽

pp. 979-987 ◽

Cited By ~ 22

Author(s):

Shane D. Kirby ◽

Scott D. Gray‐Owen ◽

Anthony B. Schryvers

Keyword(s):

Haemophilus Influenzae ◽

Binding Protein ◽

Ferric Binding Protein

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Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring

ACS Chemical Neuroscience ◽

10.1021/acschemneuro.8b00263 ◽

2018 ◽

Vol 10 (4) ◽

pp. 1970-1977

Author(s):

Gonca Bulbul ◽

Goksin Liu ◽

Namrata Rao Vithalapur ◽

Canan Atilgan ◽

Zehra Sayers ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Binding Protein ◽

Iron Binding ◽

Iron Binding Protein

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Specificity of the Synergistic Anion for Iron Binding by Ferric Binding Protein fromNeisseria gonorrhoeae†

Biochemistry ◽

10.1021/bi036143q ◽

2004 ◽

Vol 43 (28) ◽

pp. 9195-9203 ◽

Cited By ~ 10

Author(s):

Elena G. Bekker ◽

A. Louise Creagh ◽

Nooshafarin Sanaie ◽

Fumiaki Yumoto ◽

Gloria H. Y. Lau ◽

...

Keyword(s):

Binding Protein ◽

Iron Binding ◽

Ferric Binding Protein

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The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

Biochemical Journal ◽

10.1042/bj20061589 ◽

2007 ◽

Vol 403 (1) ◽

pp. 43-48 ◽

Cited By ~ 13

Author(s):

Ali G. Khan ◽

Stephen R. Shouldice ◽

Leslie W. Tari ◽

Anthony B. Schryvers

Keyword(s):

Haemophilus Influenzae ◽

Iron Transport ◽

Binding Protein ◽

Critical Role ◽

Anion Binding ◽

Iron Binding ◽

Phosphate Anion ◽

Intact Cells ◽

Terminal Domain

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.

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High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin

Biochemical Journal ◽

10.1042/bj20070110 ◽

2007 ◽

Vol 404 (2) ◽

pp. 217-225 ◽

Cited By ~ 19

Author(s):

Ali G. Khan ◽

Stephen R. Shouldice ◽

Shane D. Kirby ◽

Rong-hua Yu ◽

Leslie W. Tari ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Metal Binding ◽

Binding Assay ◽

Rank Order ◽

Binding Protein ◽

Iron Binding ◽

Wild Type ◽

Mutant Proteins ◽

High Affinity ◽

Iron Binding Protein

The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0×1018 M−1. These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.

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