The biophysical characterization of the transcription factor MntR from Mycobacterium tuberculosis and its selected mutants.

Protein MntR is a transcription factor regulating Mn2+ homeostasis in Mycobacterium tuberculosis, the causative agent of tuberculosis. A combination of computational (QM, MD, protein-DNA docking) and experimental (AUC, ITC, DSC, EPR, NMR, CD, EMSA) methods was used to characterize wild-type MntR and its selected mutants, as well as its complex with the target DNA sequence. Molecular dynamics simulations revealed that the binding of Mn2+ into the mononuclear metal-binding site is crucial for the distance and orientation of the DNA-binding helices of the protein, which is necessary for adequate DNA binding. The network of noncovalent interactions between the DNA-binding domain and the FeoA-like domain, which is important for protein structural and dynamical properties, was identified. The roles of key amino acid residues of the identified interaction network were further investigated by simulations of in silico prepared mutants. The R167A mutant was also experimentally characterized, and the results showed the importance of the interaction hub between the N-terminal domain and C-terminal domain and its influence on overall MntR properties. A novel potential DNA binding motif for MntR was identified and its interactions with the protein were computationally and experimentally described.