K182G substitution in DevR or C₈G mutation in the Dev box impairs protein-DNA interaction and abrogates DevR-mediated...

The DevR response regulator mediates adaptation of Mycobacterium tuberculosis to various signals that are likely to be encountered within the host such as hypoxia, nitric oxide, carbon monoxide and ascorbic acid. DevR is proposed as a promising target for developing drugs against dormant bacteria. It induces the expression of target genes by interacting with DNA motifs located in their promoter regions. An understanding of DNA-protein interactions is expected to facilitate the development of inhibitors targeting DevR. Only three amino acids in DevR, namely Lys179, Lys182 and Asn183, directly contact nucleotide bases in the DNA motif. The present study was designed to decipher the contribution of Lys182 in DevR function. M. tuberculosis fdxA (Rv2007c), a member of the DevR regulon, was selected for this analysis. Its transcriptional start point was mapped at -1 or -2 with respect to the putative translational start site suggesting that fdxA is expressed as a leaderless mRNA. DNase I footprinting led to the discovery of a secondary binding site and induction of the fdxA promoter is explained by the cooperative binding of DevR to two binding sites. Mutation of Lys182 lowers the DNA binding affinity of DevR and abrogates induction of fdxA and other regulon genes. Mutational analyses also highlight the singular importance of Lys182-G(13) nucleotide interaction for DevR binding and regulon induction. Our findings demonstrate that impairment of Lys182-mediated interactions alone abolishes DevR function and provide valuable insights for designing molecules that interfere with DevR-mediated dormancy adaptation.