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Deletion of the proline‐rich region of TonB disrupts formation of a 2:1 complex with FhuA, an outer membrane receptor of Escherichia coli
Author(s) -
Khursigara Cezar M.,
De Crescenzo Gregory,
Pawelek Peter D.,
Coulton James W.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051342505
Subject(s) - periplasmic space , bacterial outer membrane , biology , chemiosmosis , biophysics , escherichia coli , siderophore , inner membrane , biochemistry , crystallography , membrane , chemistry , atp synthase , gene , enzyme
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 (TonBΔ66‐100) prevents formation of the 2:1 complex. Sedimentation velocity analytical ultracentrifugation of TonBΔ66‐100 with FhuA revealed that a 1:1 TonB–FhuA complex is formed. Interactions between TonBΔ66‐100 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, TonBΔ66‐100–FhuAΔ21‐128 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.