K182G substitution in DevR or C₈G mutation in the Dev box impairs protein–DNA interaction and abrogates DevR‐mediated gene induction in Mycobacterium tuberculosis

Abstract

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–G13 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.

Essentiality of DevR/DosR Interaction with SigA for the Dormancy Survival Program in Mycobacterium tuberculosis

“…For the first time, four DevR determinants that interact with RNA polymerase have been discovered. Our findings establish DevR interaction with the transcriptional machinery as an essential step of the DevR signaling cascade in addition to the previously known steps of signal sensing, kinase autophosphorylation, transfer of the phosphosignal to DevR (35), and DevR binding to DNA, including cooperative interactions (13,27) (Fig. 6).…”

“…We assumed that the residues on the protein surface are likely to interact with RNA polymerase at the target promoter(s), and the present study was restricted to charged residues only. The C-terminal domain of DevR (PDB file name 3C3W) (11) (27). DevR C activates DevR regulon expression, albeit weakly (19), implying that this domain interacts with the transcriptional machinery.…”

Adaptation to Environmental Stimuli within the Host: Two-Component Signal Transduction Systems of Mycobacterium tuberculosis

“…EMSAs, DNase I footprinting, and gene reporter expression studies carried out thus far generally support this contention (4, 24-26, 42, 55, 116, 125, 162). In particular, DosR recognizes a fairly conserved 18/20-bp palindromic sequence that is present with some variation upstream of the transcriptional units for these genes (Table 3) (4,25,26,42,55,116,125,162). Similar sequences are also found upstream of other genes outside this regulon, although their regulation by DosR remains unclear (116,185).…”

“…Promoter regions from DosR regulon genes often contain two or more sets of recognition sequences, and cooperative binding by DosR to these sites is necessary for their full induction (24)(25)(26). Binding by DosR to its recognition sequences is mediated primarily through the C-terminal domain (176,177), and amino acid residues within this region that are important for contacting DNA have been defined and characterized (55,176). However, the N-terminal domain also regulates aspects of DosR binding and subsequent gene regulation.…”

MprA and DosR Coregulate a Mycobacterium tuberculosis Virulence Operon Encoding Rv1813c and Rv1812c

“…3A). SigC has been implicated in regulation of other DosRdependent genes (11,12,23) and may also participate in transcriptional initiation of Rv1813c. The region protected by MprA lies immediately upstream of the observed Rv1813c TSP and overlaps with a portion of the putative SigC binding sequence.…”