title: Specificity of the synergistic anion for iron binding by ferric binding protein from Neisseria gonorrhoeae.
abstract: Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Bekker
authors.forename: A Louise
authors.initials: AL
authors.lastname: Creagh
authors.forename: Nooshafarin
authors.initials: N
authors.lastname: Sanaie
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Yumoto
authors.forename: Gloria H Y
authors.initials: GH
authors.lastname: Lau
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.forename: Charles A
authors.initials: CA
authors.lastname: Haynes
authors.forename: Michael E P
authors.initials: ME
authors.lastname: Murphy
mesh_terms: D019943:Amino Acid Substitution; D000838:Anions / Q000494:pharmacology*; D004357:Drug Synergism; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017186:Titrimetry
chemical_list: D000838:Anions; D033862:Iron-Binding Proteins; D010710:Phosphates; D007501:Iron; D009571:Nitrilotriacetic Acid