Deletion of the proline-rich region of TonB disrupts formation of a 2:1 complex with FhuA, an outer membrane receptor of Escherichia coli.
TonB protein of Escherichia coli couples the electrochemical potential of the cytoplasmic membrane (CM) to active transport of iron-siderophores and vitamin B(12) across the outer membrane (OM). TonB interacts with OM receptors and transduces conformationally stored energy. Energy for transport is provided by the proton motive force through ExbB and ExbD, which form a ternary complex with TonB in the CM. TonB contains three distinct domains: an N-terminal signal/anchor sequence, a C-terminal domain, and a proline-rich region. The proline-rich region was proposed to extend TonB's structure across the periplasm, allowing it to contact spatially distant OM receptors. Having previously identified a 2:1 stoichiometry for the complex of full-length (FL) TonB and the OM receptor FhuA, we now demonstrate that deletion of the proline-rich region of TonB (TonBDelta66-100) prevents formation of the 2:1 complex. Sedimentation velocity analytical ultracentrifugation of TonBDelta66-100 with FhuA revealed that a 1:1 TonB-FhuA complex is formed. Interactions between TonBDelta66-100 and FhuA were assessed by surface plasmon resonance, and their affinities were determined to be similar to those of TonB (FL)-FhuA. Presence of the FhuA-specific siderophore ferricrocin altered neither stoichiometry nor affinity of interaction, leading to our conclusion that the proline-rich region in TonB is important in forming a 2:1 high-affinity TonB-FhuA complex in vitro. Furthermore, TonBDelta66-100-FhuADelta21-128 interactions demonstrated that the cork region of the OM receptor was also important in forming a complex. Together, these results demonstrate a novel function of the proline-rich region of TonB in mediating TonB-TonB interactions within the TonB-FhuA complex.
Khursigara
Cezar M
CM
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada H3A 2B4.
De Crescenzo
Gregory
G
Pawelek
Peter D
PD
Coulton
James W
JW
D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D011392:Proline / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; C489181:tonB protein, E coli; C069968:fhuB protein, E coli; D011392:Proline