title: Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.
abstract: The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.
authors.affiliation: Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Freed
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Lukasik
authors.forename: Arthur
authors.initials: A
authors.lastname: Sikora
authors.forename: Audrey
authors.initials: A
authors.lastname: Mokdad
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D003087:Colicins / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; C028589:FecA protein, E coli; C050900:FhuA protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; C489181:tonB protein, E coli