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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

¹ Department of Microbiology and Immunology and ² Protein–Protein Interaction Facility, Sheldon Biotechnology Center, McGill University, Montreal, Quebec, Canada H3A 2B4

(RECEIVED January 6, 2005; FINAL REVISION January 27, 2005; ACCEPTED January 27, 2005)

TonB protein of Escherichia coli couples the electrochemical potential of the cytoplasmic membrane (CM) to active transport of iron-siderophores and vitamin B₁₂ 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 (TonB66-100) prevents formation of the 2:1 complex. Sedimentation velocity analytical ultracentrifugation of TonB66-100 with FhuA revealed that a 1:1 TonB–FhuA complex is formed. Interactions between TonB66-100 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, TonB66-100–FhuA21-128 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.

Keywords: analytical ultracentrifugation; ferricrocin; FhuA; surface plasmon resonance; TonB

Abbreviations: _avg, weight-averaged apparent vbar • AUC, analytical ultracentrifugation • C8E4, n-octyltetraoxyethylene • CM, cytoplasmic membrane • CM4, carboxymethylated dextran matrix sensor chip • EDC, N-ethyl-N-(3-dimethylaminopropyl) carbodiimide-hydrochloride • Fc, ferricrocin • LDAO, lauryldimethylamine oxide • M_b, buoyant molecular mass • NHS, N-hydroxysuccinimide • NTA, Ni²⁺-nitrilotriacetic • OM, outer membrane • PDEA, 2-(2-pyridinylthio) ethanolamine • RU, resonance unit(s) • SPR, surface plasmon resonance

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051342505.

Reprint requests to: James W. Coulton, Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada H3A 2B4; e-mail: james.coulton{at}mcgill.ca; fax: (514) 398-7052.

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