Abstract
The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1–25, 58–69, 90–111, 115–132 and 157–196 and residues 35–57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.
Highlights
After its initial onset of infection in humans, the pathogen Mycobacterium tuberculosis (Mtb) becomes latent in a large number of cases, until a cellular stimulus reactivates it
Gene expression is mostly regulated at the pre-transcription level, where sigma factors play a major role
Under oxidative stress, sigH induces the expression of Mtb redox systems, such as thioredoxins trxA, trxB1, trxC and thioredoxin reductase trxB2, whereas, it induces the expression of the hsp70 and clpB genes under heat stress [4,8,12]
Summary
After its initial onset of infection in humans, the pathogen Mycobacterium tuberculosis (Mtb) becomes latent in a large number of cases, until a cellular stimulus reactivates it. It is assumed that since Mtb RshA has a ZAS motif and its closest homologue RsrA of S. coelicolor is a zinc binding protein, where Zn ion plays a major role, RshA has been proposed to be a zinc binding protein and its interaction with SigH is regulated by zinc. Formation of the holoenzyme would enable transcription and SigH complexed with RNA polymerase would bind to its own promoter and regulate transcription [3] This could be an alternate mechanism of regulation of SigH in Mtb which might be important at some stages of its survival in its host. No structural map of the RshA-SigH complex exists It is unknown if the mechanism of RshA-SigH interactions are identical to that of the RsrA and SigR interaction and probe details on the importance of the metal ion cluster.
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