Interaction of Mycobacterium tuberculosis RshA and SigH is mediated by salt bridges

PLoS One. 2012;7(8):e43676. doi: 10.1371/journal.pone.0043676. Epub 2012 Aug 24.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / metabolism
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / metabolism
  • Sigma Factor / genetics*
  • Sigma Factor / metabolism
  • Transcription, Genetic

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Intracellular Signaling Peptides and Proteins
  • RshA protein, Mycobacterium tuberculosis
  • SigH protein, bacteria
  • Sigma Factor

Grant support

KS acknowledges the funding support from the Ministry of Education (Faculty Research Council, R154-000-439-112) and Biomedical Research Council (R154-000-424-305), Singapore. PA acknowledges the funding support from the SIP-10 project of Council of Scientific and Industrial Research (CSIR) for this project and for SS. Both SKG and MSA were recipients of the CSIR, India Senior Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.