Kinetic analyses reveal multiple steps in forming TonB-FhuA complexes from Escherichia coli

Biochemistry. 2005 Mar 8;44(9):3441-53. doi: 10.1021/bi047882p.


FhuA, an outer membrane receptor of Escherichia coli, facilitates transport of hydroxamate siderophores and siderophore-antibiotic conjugates. The cytoplasmic membrane complex TonB-ExbB-ExbD provides energy for transport via the proton motive force. This energy is transduced by protein-protein interactions between TonB and FhuA, but the molecular determinants of these interactions remain uncharacterized. Our analyses of FhuA and two recombinant TonB species by surface plasmon resonance revealed that TonB undergoes a kinetically limiting rearrangement upon initial interaction with FhuA: an intermediate TonB-FhuA complex of 1:1 stoichiometry was detected. The intermediate then recruits a second TonB protein. Addition of ferricrocin, a FhuA-specific ligand, enhanced amounts of the 2:1 complex but was not essential for its formation. To assess the role of the cork domain of FhuA in forming a 2:1 TonB-FhuA complex, we tested a FhuA deletion (residues 21-128) for its ability to interact with TonB. Analytical ultracentrifugation demonstrated that deletion of this region of the cork domain resulted in a 1:1 complex. Furthermore, the high-affinity 2:1 complex requires the N-terminal region of TonB. Together these in vitro experiments establish that TonB-FhuA interactions require sequential steps of kinetically limiting rearrangements. Additionally, domains that contribute to complex formation were identified in TonB and in FhuA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cysteine / genetics
  • Cytoplasm / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Ferrichrome / analogs & derivatives*
  • Ferrichrome / metabolism
  • Kinetics
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Models, Chemical
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism*
  • Mutagenesis, Site-Directed
  • Protein Interaction Mapping
  • Protein Structure, Secondary / genetics
  • Protein Structure, Tertiary / genetics
  • Receptors, Virus / chemistry
  • Receptors, Virus / genetics
  • Receptors, Virus / metabolism*
  • Sequence Deletion
  • Surface Plasmon Resonance
  • Thermodynamics


  • Bacterial Outer Membrane Proteins
  • Escherichia coli Proteins
  • FhuA protein, E coli
  • Membrane Proteins
  • Multiprotein Complexes
  • Receptors, Virus
  • tonB protein, E coli
  • Ferrichrome
  • ferricrocin
  • Cysteine