title: Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum.
abstract: In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121-206) has an alphabetabetaalphabeta structure, whereas residues 76-120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the beta4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 A (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the beta4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.
authors.affiliation: Department of Molecular Microbiology and Immunology L-220, Oregon Health and Science University, Portland, OR 97239, USA.
authors.forename: Claudia S
authors.initials: CS
authors.lastname: López
authors.forename: R Sean
authors.initials: RS
authors.lastname: Peacock
authors.forename: Jorge H
authors.initials: JH
authors.lastname: Crosa
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005393:Fish Diseases / Q000382:microbiology*; D017353:Gene Deletion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D055672:Static Electricity; D014733:Vibrio / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010455:Peptides; D011994:Recombinant Proteins; C057616:tonB protein, Bacteria; C071990:FatA protein, bacteria; C049469:anguibactin