title: Solution structure and peptide binding studies of the C-terminal src homology 3-like domain of the diphtheria toxin repressor protein.
abstract: The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five beta-strands and three helices arranged into a partially orthogonal, two-sheet beta-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in beta-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: G
authors.initials: G
authors.lastname: Wang
authors.forename: G P
authors.initials: GP
authors.lastname: Wylie
authors.forename: P D
authors.initials: PD
authors.lastname: Twigg
authors.forename: D L
authors.initials: DL
authors.lastname: Caspar
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
authors.forename: T M
authors.initials: TM
authors.lastname: Logan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D018909:src Homology Domains
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012996:Solutions; D007501:Iron