title: Interactions between TonB from Escherichia coli and the periplasmic protein FhuD.
abstract: For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.
authors.affiliation: Department of Microbiology and Immunology, and Sheldon Biotechnology Centre, McGill University, Montreal, Quebec H3A 2B4, Canada.
authors.forename: David M
authors.initials: DM
authors.lastname: Carter
authors.forename: Isabelle R
authors.initials: IR
authors.lastname: Miousse
authors.forename: Jean-Nicolas
authors.initials: JN
authors.lastname: Gagnon
authors.forename: Eric
authors.initials: E
authors.lastname: Martinez
authors.forename: Abigail
authors.initials: A
authors.lastname: Clements
authors.forename: Jongchan
authors.initials: J
authors.lastname: Lee
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hancock
authors.forename: Hubert
authors.initials: H
authors.lastname: Gagnon
authors.forename: Peter D
authors.initials: PD
authors.lastname: Pawelek
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019151:Peptide Library; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D019151:Peptide Library; D033902:Periplasmic Binding Proteins; C489181:tonB protein, E coli; C069969:fhuD protein, E coli