title: Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport.
abstract: Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a β-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the β-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.
authors.affiliation: Department of Chemistry, Duke University, Durham, NC-27708-0346, USA.
authors.forename: Sambuddha
authors.initials: S
authors.lastname: Banerjee
authors.forename: Claire J Parker
authors.initials: CJ
authors.lastname: Siburt
authors.forename: Shreni
authors.initials: S
authors.lastname: Mistry
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Noto
authors.forename: Patrick
authors.initials: P
authors.lastname: DeArmond
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Lambert
authors.forename: Cynthia N
authors.initials: CN
authors.lastname: Cornelissen
authors.forename: Alvin L
authors.initials: AL
authors.lastname: Crumbliss
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002942:Circular Dichroism; D006069:Gonorrhea / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism; D033881:Transferrin-Binding Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; D014168:Transferrin; D033881:Transferrin-Binding Protein A; D007501:Iron