title: FecB, a periplasmic ferric-citrate transporter from E. coli, can bind different forms of ferric-citrate as well as a wide variety of metal-free and metal-loaded tricarboxylic acids.
abstract: The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low μM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with μM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. vogel@ucalgary.ca.
authors.forename: Sambuddha
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. vogel@ucalgary.ca.
authors.forename: Subrata
authors.initials: S
authors.lastname: Paul
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. vogel@ucalgary.ca.
authors.forename: Leonard T
authors.initials: LT
authors.lastname: Nguyen
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. vogel@ucalgary.ca.
authors.forename: Byron C H
authors.initials: BC
authors.lastname: Chu
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. vogel@ucalgary.ca.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D019343:Citric Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D016623:Ion Pumps / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism*; D009587:Nitrogen Isotopes; D019897:Periplasm / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D014233:Tricarboxylic Acids / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D016623:Ion Pumps; D008024:Ligands; D008670:Metals; D009587:Nitrogen Isotopes; D014233:Tricarboxylic Acids; C069970:FecB protein, E coli; D019343:Citric Acid; C025314:ferric citrate