title: Insight from TonB hybrid proteins into the mechanism of iron transport through the outer membrane.
abstract: We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) 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 (FepADelta3-17, 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 to E. coli YcfS, 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA.
authors.forename: Wallace A
authors.initials: WA
authors.lastname: Kaserer
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.forename: Qiaobin
authors.initials: Q
authors.lastname: Xiao
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Scott
authors.forename: Matthew
authors.initials: M
authors.lastname: Bauler
authors.forename: Daniel
authors.initials: D
authors.lastname: Copeland
authors.forename: Salete M C
authors.initials: SM
authors.lastname: Newton
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Klebba
mesh_terms: D000595:Amino Acid Sequence; D001693:Biological Transport, Active / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C489181:tonB protein, E coli; D007501:Iron