Interactions of Metal-ions with a Novel Manganese-regulatory-protein from Porphyromonas gingivalis

Objective: Porphyromonas gingivalis is an anaerobic Gram-negative oral pathogen which is highly associated with the progression of chronic periodontitis. P. gingivalis is known to utilize manganese in its resistance to oxidative stress. A protein encoded in the same operon as the novel P. gingivalis manganese transporter FeoB2 has been predicted to be a manganese responsive transcriptional regulator (Pg-MntR) from the DtxR family. Pg-MntR has an additional FeoA-like domain of unknown function fused to the C-terminus. To elucidate the effect of metal ions on the conformation of Pg-MntR, a recombinant protein has been expressed and purified and several biophysical studies have been performed. Methods: Pg-MntR was purified in the presence and absence of EDTA for determination of bound metal using inductively coupled plasma mass spectrometry. Circular dichroism (CD) spectroscopy was used to determine the thermostability of Pg-MntR and the effect of metal on the secondary structure in the presence of divalent metal ions (Zn²⁺, Fe²⁺ and Mn²⁺). Conformational changes of Pg-MntR upon addition of various metal ions were investigated using the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS). Results: Heating of Pg-MntR in the absence of metal ions resulted in a clear, irreversible melting transition at 55°C. The addition of metal ions induced changes in the CD spectra of Pg-MntR, indicating an effect of metal ions on the secondary structure of the protein. The presence of Pg-MntR enhanced the fluorescence at 490 nm emitted by ANS and this enhanced fluorescence decreased with addition of metal ions, indicative of a conformational change of Pg-MntR. Conclusion: Zn²⁺, Fe²⁺ and Mn²⁺ interact with Pg-MntR and induce conformational changes that may be important for activation of Pg-MntR to bind DNA as a transcriptional regulator.