TonB interacts with BtuF, the Escherichia coli periplasmic binding protein for cyanocobalamin.
By its direct contact with outer membrane receptor BtuB, the cytoplasmic membrane transducer TonB delivers energy that mediates cyanocobalamin uptake in Escherichia coli. This activity has been generally proposed to be the role of TonB in cyanocobalamin uptake. We now report the discovery and characterization of interactions between TonB and periplasmic binding protein BtuF. Phage display experiments predicted interaction between TonB and BtuF, identifying potential binding residues on each protein. Dynamic light scattering experiments measured a complex of 55 kDa, consistent with a TonB-BtuF heterodimer. The hydrodynamic radius of the complex was unchanged in the presence of cyanocobalamin. Surface plasmon resonance measured TonB-BtuF interaction kinetics that were independent of cyanocobalamin and that deviated from a simple binding model. Binding isotherms from intrinsic fluorescence suggested a multifaceted interaction that was independent of cyanocobalamin. In addition, the presence of TonB did not abrogate subsequent binding of cyanocobalamin by BtuF. Taken together, these data support a previously proposed model wherein TonB serves as a scaffold to optimally position BtuF for initial binding of cyanocobalamin and for its subsequent release. These results substantiate a diverse role for TonB with its multiple protein-protein interactions in bacterial nutrient uptake systems.
James
Karron J
KJ
Department of Microbiology and Immunology, McGill University, 3775 University Street,Montreal, Quebec, Canada H3A 2B4.
Hancock
Mark A
MA
Gagnon
Jean-Nicolas
JN
Coulton
James W
JW
D000595:Amino Acid Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; D011956:Receptors, Cell Surface; C446048:btuF protein, E coli; C489181:tonB protein, E coli; C069969:fhuD protein, E coli; D014805:Vitamin B 12