title: Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue.
abstract: PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Lianyi
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Butler
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Hasnah S G
authors.initials: HS
authors.lastname: Khan
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Dashper
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Seers
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Veith
authors.affiliation: Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Zhang
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D008345:Manganese