Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity

Nature Structural & Molecular Biology volume 20, pages 740–747 (2013)Cite this article

Subjects

Abstract

IscR from Escherichia coli is an unusual metalloregulator in that both apo and iron sulfur (Fe-S)-IscR regulate transcription and exhibit different DNA binding specificities. Here, we report structural and biochemical studies of IscR suggesting that remodeling of the protein-DNA interface upon Fe-S ligation broadens the DNA binding specificity of IscR from binding the type 2 motif only to both type 1 and type 2 motifs. Analysis of an apo-IscR variant with relaxed target-site discrimination identified a key residue in wild-type apo-IscR that, we propose, makes unfavorable interactions with a type 1 motif. Upon Fe-S binding, these interactions are apparently removed, thereby allowing holo-IscR to bind both type 1 and type 2 motifs. These data suggest a unique mechanism of ligand-mediated DNA site recognition, whereby metallocluster ligation relocates a protein-specificity determinant to expand DNA target-site selection, allowing a broader transcriptomic response by holo-IscR.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overall structure of IscR-3CA bound to the hya promoter.
Figure 2: IscR-3CA-hya DNA interactions.
Figure 3: IscR-3CA binding to the hya promoter involves only minor conformational changes.
Figure 4: Elimination of the Glu43 carboxyl group of apo-IscR broadens the DNA binding specificity to include type 1 sites.
Figure 5: Apo-IscR does not bind a type 1 site containing symmetrical cytosines at positions 7 and 8 of each half-site.
Figure 6: Model for IscR discrimination between type 1 and type 2 motifs.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Johnson, D.C., Dean, D.R., Smith, A.D. & Johnson, M.K. Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281 (2005).

    Article  CAS  Google Scholar 

  2. Fleischhacker, A.S. & Kiley, P.J. Iron-containing transcription factors and their roles as sensors. Curr. Opin. Chem. Biol. 15, 335–341 (2011).

    Article  CAS  Google Scholar 

  3. Crack, J.C., Green, J., Thomson, A.J. & Le Brun, N.E. Iron-sulfur cluster sensor-regulators. Curr. Opin. Chem. Biol. 16, 35–44 (2012).

    Article  CAS  Google Scholar 

  4. Guerra, A.J. & Giedroc, D.P. Metal site occupancy and allosteric switching in bacterial metal sensor proteins. Arch. Biochem. Biophys. 519, 210–222 (2012).

    Article  CAS  Google Scholar 

  5. Fleischhacker, A.S. et al. Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR. Biochemistry 51, 4453–4462 (2012).

    Article  CAS  Google Scholar 

  6. Nesbit, A.D., Giel, J.L., Rose, J.C. & Kiley, P.J. Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation. J. Mol. Biol. 387, 28–41 (2009).

    Article  CAS  Google Scholar 

  7. Yeo, W.S., Lee, J.H., Lee, K.C. & Roe, J.H. IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins. Mol. Microbiol. 61, 206–218 (2006).

    Article  CAS  Google Scholar 

  8. Giel, J.L. et al. Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli. Mol. Microbiol. 87, 478–492 (2013).

    Article  CAS  Google Scholar 

  9. Rodionov, D.A., Gelfand, M.S., Todd, J.D., Curson, A.R. & Johnston, A.W. Computational reconstruction of iron- and manganese-responsive transcriptional networks in α-proteobacteria. PLoS Comput. Biol. 2, e163 (2006).

    Article  Google Scholar 

  10. Shepard, W. et al. Insights into the Rrf2 repressor family–the structure of CymR, the global cysteine regulator of Bacillus subtilis. FEBS J. 278, 2689–2701 (2011).

    Article  CAS  Google Scholar 

  11. Schwartz, C.J. et al. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98, 14895–14900 (2001).

    Article  CAS  Google Scholar 

  12. Giel, J.L., Rodionov, D., Liu, M., Blattner, F.R. & Kiley, P.J. IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol. Microbiol. 60, 1058–1075 (2006).

    Article  CAS  Google Scholar 

  13. Lazazzera, B.A., Beinert, H., Khoroshilova, N., Kennedy, M.C. & Kiley, P.J. DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen. J. Biol. Chem. 271, 2762–2768 (1996).

    Article  CAS  Google Scholar 

  14. Hidalgo, E. & Demple, B. An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein. EMBO J. 13, 138–146 (1994).

    Article  CAS  Google Scholar 

  15. Gaudu, P. & Weiss, B. SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form. Proc. Natl. Acad. Sci. USA 93, 10094–10098 (1996).

    Article  CAS  Google Scholar 

  16. Ebright, R.H. et al. Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP. Proc. Natl. Acad. Sci. USA 84, 6083–6087 (1987).

    Article  CAS  Google Scholar 

  17. Komori, H. et al. Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6-Å resolution. EMBO J. 18, 4597–4607 (1999).

    Article  CAS  Google Scholar 

  18. MacPherson, S., Larochelle, M. & Turcotte, B. A fungal family of transcriptional regulators: the zinc cluster proteins. Microbiol. Mol. Biol. Rev. 70, 583–604 (2006).

    Article  CAS  Google Scholar 

  19. Reeves, R. & Nissen, M.S. The A•T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J. Biol. Chem. 265, 8573–8582 (1990).

    CAS  PubMed  Google Scholar 

  20. Rohs, R. et al. Origins of specificity in protein-DNA recognition. Annu. Rev. Biochem. 79, 233–269 (2010).

    Article  CAS  Google Scholar 

  21. Rohs, R. et al. The role of DNA shape in protein-DNA recognition. Nature 461, 1248–1253 (2009).

    Article  CAS  Google Scholar 

  22. Haran, T.E. & Mohanty, U. The unique structure of A-tracts and intrinsic DNA bending. Q. Rev. Biophys. 42, 41–81 (2009).

    Article  CAS  Google Scholar 

  23. Coulocheri, S.A., Pigis, D.G., Papavassiliou, K.A. & Papavassiliou, A.G. Hydrogen bonds in protein-DNA complexes: where geometry meets plasticity. Biochimie 89, 1291–1303 (2007).

    Article  CAS  Google Scholar 

  24. Ding, H., Hidalgo, E. & Demple, B. The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor. J. Biol. Chem. 271, 33173–33175 (1996).

    Article  CAS  Google Scholar 

  25. Watanabe, S., Kita, A., Kobayashi, K. & Miki, K. Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA. Proc. Natl. Acad. Sci. USA 105, 4121–4126 (2008).

    Article  CAS  Google Scholar 

  26. Imlay, J.A. Iron-sulphur clusters and the problem with oxygen. Mol. Microbiol. 59, 1073–1082 (2006).

    Article  Google Scholar 

  27. Nesbit, A.D., Fleischhacker, A.S., Teter, S.J. & Kiley, P.J. ArcA and AppY antagonize IscR repression of hydrogenase 1 expression under anaerobic conditions, revealing a novel mode of O2 regulation of gene expression in Escherichia coli. J. Bacteriol. 194, 6892–6899 (2012).

    Article  CAS  Google Scholar 

  28. Shin, J.-H. et al. Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur. Proc. Natl. Acad. Sci. USA 108, 5045–5050 (2011).

    Article  CAS  Google Scholar 

  29. Ohmura, T., Ueda, T., Hashimoto, Y. & Imoto, T. Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme. Protein Eng. 14, 421–425 (2001).

    Article  CAS  Google Scholar 

  30. Leahy, D.J., Erickson, H.P., Aukhil, I., Joshi, P. & Hendrickson, W.A. Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine. Proteins 19, 48–54 (1994).

    Article  CAS  Google Scholar 

  31. Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  Google Scholar 

  32. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  34. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  35. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  36. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  38. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  39. Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

    Article  Google Scholar 

  40. McNicholas, S., Potterton, E., Wilson, K.S. & Noble, M.E. Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr. D Biol. Crystallogr. 67, 386–394 (2011).

    Article  CAS  Google Scholar 

  41. Fischer, D.S. & Price, D.C. A simple serum iron method using the new sensitive chromogen tripyridyl-s-triazine. Clin. Chem. 10, 21–31 (1964).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the organizers and instructors of the 2012 CCP4 APS school (attended by S.R.) for providing valuable insights into analyzing the data. Structural data were collected at the Berkeley Center for Structural Biology (BCSB) beamlines at Advanced Light Source (ALS) and at the Northeastern Collaborative Access Team (NE-CAT) beamlines at Advanced Photon Source (APS). BCSB is supported in part by the NIH, the National Institute of General Medical Sciences and the Howard Hughes Medical Institute. The ALS is supported by the Director, Office of Science and the Office of Basic Energy Sciences of the US Department of Energy (DOE) under contract no. DE-AC02-05CH11231. The NE-CAT beamlines are supported by grants from the National Center for Research Resources (5P41RR015301-10) and the National Institute of General Medical Sciences (8 P41 GM103403-10). Use of the APS, operated for the DOE Office of Science by Argonne National Laboratory, was supported by the DOE under contract no. DE-AC02-06CH11357. This work was funded by grants from the US National Institutes of Health (NIH; GM045844 to P.J.K.) and The Methodist Hospital Research Institute to K.J.P.

Author information

Author notes

  1. Senapathy Rajagopalan and Sarah J Teter: These authors contributed equally to this work.

Authors and Affiliations

  1. Genomic Medicine Program, The Methodist Hospital Research Institute, Houston, Texas, USA

    Senapathy Rajagopalan & Kevin J Phillips

  2. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Sarah J Teter & Patricia J Kiley

  3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Petrus H Zwart

  4. Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA

    Richard G Brennan

  5. Diabetes Research Program, The Methodist Hospital Research Institute, Houston, Texas, USA

    Kevin J Phillips

  6. Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA

    Kevin J Phillips

Authors
  1. Senapathy Rajagopalan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah J Teter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Petrus H Zwart
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard G Brennan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kevin J Phillips
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patricia J Kiley
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.J.K., K.J.P., S.J.T., S.R. and R.G.B. designed the experiments. S.R., S.J.T. and P.H.Z. performed the experiments. P.J.K., K.J.P., S.J.T. and S.R. wrote the paper.

Corresponding authors

Correspondence to Kevin J Phillips or Patricia J Kiley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 15284 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rajagopalan, S., Teter, S., Zwart, P. et al. Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity. Nat Struct Mol Biol 20, 740–747 (2013). https://doi.org/10.1038/nsmb.2568

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2568

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing