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.

Repair of Iron Center Proteins—A Different Class of Hemerythrin-like Proteins

“…Hence it was of relevance to determine the affinity of the wild type YtfE to manganese and compare it with that of iron. This study was done by analyzing the quenching of the intrinsic fluorescence intensity [49] of YtfE upon the addition of Mn 2+ or Fe 2+ , and determination of the binding constants for each metal. Both the apoproteins of the YtfE wild type and the YtfE-E159L variant exhibited intrinsic fluorescence emission spectra, with maxima intensities at ~326 nm.…”

“…Apo-YtfE and apo-E159L variant protein solutions (5 µM), were prepared in 50 mM HEPES (pH 6.8) with 150 mM NaCl under reducing conditions (with 2 mM Tris (2-carboxyethyl)phosphine, TCEP). Samples were excited at 280 nm and fluorescence spectra were collected in the 290 to 450 nm range [49]. The maximum fluorescence intensity occurred at ~326 nm.…”