title: High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin.
abstract: 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 >2.0x10(18) 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.
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada T2N 4N1.
authors.forename: Ali G
authors.initials: AG
authors.lastname: Khan
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Shouldice
authors.forename: Shane D
authors.initials: SD
authors.lastname: Kirby
authors.forename: Rong-hua
authors.initials: RH
authors.lastname: Yu
authors.forename: Leslie W
authors.initials: LW
authors.lastname: Tari
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Schryvers
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006193:Haemophilus influenzae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D014168:Transferrin; D007501:Iron