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Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges
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-transl...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427169/ https://www.ncbi.nlm.nih.gov/pubmed/22937074 http://dx.doi.org/10.1371/journal.pone.0043676 |
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author | Kumar, Shiva Badireddy, Suguna Pal, Kuntal Sharma, Shikha Arora, Chandni Garg, Saurabh K. Alam, Mohamed Suhail Agrawal, Pushpa Anand, Ganesh Srinivasan Swaminathan, Kunchithapadam |
author_facet | Kumar, Shiva Badireddy, Suguna Pal, Kuntal Sharma, Shikha Arora, Chandni Garg, Saurabh K. Alam, Mohamed Suhail Agrawal, Pushpa Anand, Ganesh Srinivasan Swaminathan, Kunchithapadam |
author_sort | Kumar, Shiva |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3427169 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34271692012-08-30 Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges Kumar, Shiva Badireddy, Suguna Pal, Kuntal Sharma, Shikha Arora, Chandni Garg, Saurabh K. Alam, Mohamed Suhail Agrawal, Pushpa Anand, Ganesh Srinivasan Swaminathan, Kunchithapadam PLoS One Research Article 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. Public Library of Science 2012-08-24 /pmc/articles/PMC3427169/ /pubmed/22937074 http://dx.doi.org/10.1371/journal.pone.0043676 Text en © 2012 Kumar et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kumar, Shiva Badireddy, Suguna Pal, Kuntal Sharma, Shikha Arora, Chandni Garg, Saurabh K. Alam, Mohamed Suhail Agrawal, Pushpa Anand, Ganesh Srinivasan Swaminathan, Kunchithapadam Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title | Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title_full | Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title_fullStr | Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title_full_unstemmed | Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title_short | Interaction of Mycobacterium tuberculosis RshA and SigH Is Mediated by Salt Bridges |
title_sort | interaction of mycobacterium tuberculosis rsha and sigh is mediated by salt bridges |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427169/ https://www.ncbi.nlm.nih.gov/pubmed/22937074 http://dx.doi.org/10.1371/journal.pone.0043676 |
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