Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane
- 1 June 2008
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 190 (11), 4001-4016
- https://doi.org/10.1128/jb.00135-08
Abstract
We created hybrid proteins to study the functions of TonB. We first fused the portion ofEscherichia coli tonBthat encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream fromE. coli malE(MalE-TonB69C). Production of MalE-TonB69C intonB+bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus toE. coliYcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.Keywords
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