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CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator

Nature Chemical Biology volume 3, pages 60–68 (2007)Cite this article

Abstract

Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S2N) Cu(I) complex. The 2.6-Å crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65′ and His61′ in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.

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Figure 1: Expression of a novel operon in Mtb after exposure to high concentrations of copper.
Figure 2: Schematic representation of the organization of the Mtb cso operon and a multiple sequence alignment of CsoR homologs in bacteria.
Figure 3: Expression from the cso promoter in M.smegmatis is specifically induced by addition of copper salts to the growth medium.
Figure 4: CsoR is a Cu(I)-binding protein that is saturable with 1 monomer mol equiv. of Cu(I).
Figure 5: Crystallographic structure of Mtb CsoR4–88.
Figure 6: XAS of Cu-CsoR.

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References

  1. Taylor, A.B., Stoj, C.S., Ziegler, L., Kosman, D.J. & Hart, P.J. The copper-iron connection in biology: structure of the metallo-oxidase Fet3p. Proc. Natl. Acad. Sci. USA 102, 15459–15464 (2005).

    Article  CAS  Google Scholar 

  2. Dong, J. et al. Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry 42, 2768–2773 (2003).

    Article  CAS  Google Scholar 

  3. Rasia, R.M. et al. Structural characterization of copper(II) binding to alpha-synuclein: insights into the bioinorganic chemistry of Parkinson's disease. Proc. Natl. Acad. Sci. USA 102, 4294–4299 (2005).

    Article  CAS  Google Scholar 

  4. Bocharova, O.V., Breydo, L., Salnikov, V.V. & Baskakov, I.V. Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils. Biochemistry 44, 6776–6787 (2005).

    Article  CAS  Google Scholar 

  5. Eakin, C.M., Berman, A.J. & Miranker, A.D. A native to amyloidogenic transition regulated by a backbone trigger. Nat. Struct. Mol. Biol. 13, 202–208 (2006).

    Article  CAS  Google Scholar 

  6. Rae, T.D., Schmidt, P.J., Pufahl, R.A., Culotta, V.C. & O'Halloran, T.V. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284, 805–808 (1999).

    Article  CAS  Google Scholar 

  7. Brown, K.R. et al. Structures of the cuprous-thiolate clusters of the Mac1 and Ace1 transcriptional activators. Biochemistry 41, 6469–6476 (2002).

    Article  CAS  Google Scholar 

  8. Beaudoin, J., Mercier, A., Langlois, R. & Labbe, S. The Schizosaccharomyces pombe Cuf1 is composed of functional modules from two distinct classes of copper metalloregulatory transcription factors. J. Biol. Chem. 278, 14565–14577 (2003).

    Article  CAS  Google Scholar 

  9. Egli, D. et al. Knockout of 'metal-responsive transcription factor' MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis. EMBO J. 22, 100–108 (2003).

    Article  CAS  Google Scholar 

  10. Petris, M.J., Smith, K., Lee, J. & Thiele, D.J. Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1. J. Biol. Chem. 278, 9639–9646 (2003).

    Article  CAS  Google Scholar 

  11. Outten, F.W., Outten, C.E., Hale, J. & O'Halloran, T.V. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. J. Biol. Chem. 275, 31024–31029 (2000).

    Article  CAS  Google Scholar 

  12. Solioz, M. & Stoyanov, J.V. Copper homeostasis in Enterococcus hirae. FEMS Microbiol. Rev. 27, 183–195 (2003).

    Article  CAS  Google Scholar 

  13. Rensing, C. & Grass, G. Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiol. Rev. 27, 197–213 (2003).

    Article  CAS  Google Scholar 

  14. Skaar, E.P., Humayun, M., Bae, T., DeBord, K.L. & Schneewind, O. Iron-source preference of Staphylococcus aureus infections. Science 305, 1626–1628 (2004).

    Article  CAS  Google Scholar 

  15. Agranoff, D. & Krishna, S. Metal ion transport and regulation in Mycobacterium tuberculosis. Front. Biosci. 9, 2996–3006 (2004).

    Article  CAS  Google Scholar 

  16. Spagnolo, L. et al. Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional copper-containing enzyme lacking zinc in the active site. J. Biol. Chem. 279, 33447–33455 (2004).

    Article  CAS  Google Scholar 

  17. Francis, M.S. & Thomas, C.J. Mutants in the CtpA copper transporting P-type ATPase reduce virulence of Listeria monocytogenes. Microb. Pathog. 22, 67–78 (1997).

    Article  CAS  Google Scholar 

  18. Mitrakul, K., Loo, C.Y., Hughes, C.V. & Ganeshkumar, N. Role of a Streptococcus gordonii copper-transport operon, copYAZ, in biofilm detachment. Oral Microbiol. Immunol. 19, 395–402 (2004).

    Article  CAS  Google Scholar 

  19. Wagner, D. et al. Changes of the phagosomal elemental concentrations by Mycobacterium tuberculosis Mramp. Microbiology 151, 323–332 (2005).

    Article  CAS  Google Scholar 

  20. Strausak, D. & Solioz, M. CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases. J. Biol. Chem. 272, 8932–8936 (1997).

    Article  CAS  Google Scholar 

  21. Stoyanov, J.V., Hobman, J.L. & Brown, N.L. CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Mol. Microbiol. 39, 502–511 (2001).

    Article  CAS  Google Scholar 

  22. Gaballa, A., Cao, M. & Helmann, J.D. Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon. Microbiology 149, 3413–3421 (2003).

    Article  CAS  Google Scholar 

  23. Portmann, R., Poulsen, K.R., Wimmer, R. & Solioz, M. CopY-like copper inducible repressors are putative 'winged helix' proteins. Biometals 19, 61–70 (2006).

    Article  CAS  Google Scholar 

  24. Brown, N.L., Stoyanov, J.V., Kidd, S.P. & Hobman, J.L. The MerR family of transcriptional regulators. FEMS Microbiol. Rev. 27, 145–164 (2003).

    Article  CAS  Google Scholar 

  25. Talaat, A.M., Lyons, R., Howard, S.T. & Johnston, S.A. The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc. Natl. Acad. Sci. USA 101, 4602–4607 (2004).

    Article  CAS  Google Scholar 

  26. Palmgren, M.G. & Axelsen, K.B. Evolution of P-type ATPases. Biochim. Biophys. Acta 1365, 37–45 (1998).

    Article  CAS  Google Scholar 

  27. Graham, J.E. & Clark-Curtiss, J.E. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc. Natl. Acad. Sci. USA 96, 11554–11559 (1999).

    Article  CAS  Google Scholar 

  28. Schnappinger, D. et al. Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J. Exp. Med. 198, 693–704 (2003).

    Article  CAS  Google Scholar 

  29. Talaat, A.M. et al. Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis. Nucleic Acids Res. 30, e104 (2002).

    Article  Google Scholar 

  30. Cavet, J.S., Graham, A.I., Meng, W. & Robinson, N.J. A cadmium-lead-sensing ArsR-SmtB repressor with novel sensory sites. Complementary metal discrimination by NmtR and CmtR in a common cytosol. J. Biol. Chem. 278, 44560–44566 (2003).

    Article  CAS  Google Scholar 

  31. Timm, J., Lim, E.M. & Gicquel, B. Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. J. Bacteriol. 176, 6749–6753 (1994).

    Article  CAS  Google Scholar 

  32. Liu, T. et al. A novel cyanobacterial SmtB/ArsR family repressor regulates the expression of a CPx-ATPase and a metallothionein in response to both Cu(I)/Ag(I) and Zn(II)/Cd(II). J. Biol. Chem. 279, 17810–17818 (2004).

    Article  CAS  Google Scholar 

  33. Liu, T., Golden, J.W. & Giedroc, D.P. A zinc(II)/lead(II)/cadmium(II)-inducible operon from the cyanobacterium Anabaena is regulated by AztR, an α3N ArsR/SmtB metalloregulator. Biochemistry 44, 8673–8683 (2005).

    Article  CAS  Google Scholar 

  34. Graden, J.A. et al. Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1. Biochemistry 35, 14583–14589 (1996).

    Article  CAS  Google Scholar 

  35. Xiao, Z., Loughlin, F., George, G.N., Howlett, G.J. & Wedd, A.G. C-terminal domain of the membrane copper transporter Ctr1 from Saccharomyces cerevisiae binds four Cu(I) ions as a cuprous-thiolate polynuclear cluster: sub-femtomolar Cu(I) affinity of three proteins involved in copper trafficking. J. Am. Chem. Soc. 126, 3081–3090 (2004).

    Article  CAS  Google Scholar 

  36. Chen, K., Yuldasheva, S., Penner-Hahn, J.E. & O'Halloran, T.V. An atypical linear Cu(I)-S2 center constitutes the high-affinity metal-sensing site in the CueR metalloregulatory protein. J. Am. Chem. Soc. 125, 12088–12089 (2003).

    Article  CAS  Google Scholar 

  37. Changela, A. et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387 (2003).

    Article  CAS  Google Scholar 

  38. Wheeler, D.L. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 28, 10–14 (2000).

    Article  CAS  Google Scholar 

  39. Tottey, S. et al. A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition. J. Biol. Chem. 277, 5490–5497 (2002).

    Article  Google Scholar 

  40. Heldwein, E.E. & Brennan, R.G. Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409, 378–382 (2001).

    Article  CAS  Google Scholar 

  41. Schumacher, M.A. et al. Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR. EMBO J. 21, 1210–1218 (2002).

    Article  CAS  Google Scholar 

  42. Herring, C.D. & Blattner, F.R. Global transcriptional effects of a suppressor tRNA and the inactivation of the regulator frmR. J. Bacteriol. 186, 6714–6720 (2004).

    Article  CAS  Google Scholar 

  43. Iwig, J.S., Rowe, J.L. & Chivers, P.T. Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. Mol. Microbiol. 62, 252–262 (2006).

    Article  CAS  Google Scholar 

  44. Pennella, M.A. & Giedroc, D.P. Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators. Biometals 18, 413–428 (2005).

    Article  CAS  Google Scholar 

  45. Chao, J.A. et al. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 12, 952–957 (2005).

    Article  CAS  Google Scholar 

  46. Predki, P.F., Nayak, L.M., Gottlieb, M.B. & Regan, L. Dissecting RNA-protein interactions: RNA-RNA recognition by Rop. Cell 80, 41–50 (1995).

    Article  CAS  Google Scholar 

  47. D'Orazio, M. et al. Lipid modification of the Cu,Zn superoxide dismutase from Mycobacterium tuberculosis. Biochem. J. 359, 17–22 (2001).

    Article  CAS  Google Scholar 

  48. Domenech, P., Pym, A.S., Cellier, M., Barry, C.E., III & Cole, S.T. Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis. FEMS Microbiol. Lett. 207, 81–86 (2002).

    Article  CAS  Google Scholar 

  49. Battistoni, A. Role of prokaryotic Cu,Zn superoxide dismutase in pathogenesis. Biochem. Soc. Trans. 31, 1326–1329 (2003).

    Article  CAS  Google Scholar 

  50. Cooper, J.B. et al. X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 angstroms resolution reveals novel dimer-dimer interactions. J. Mol. Biol. 246, 531–544 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Rehr, J.J., Mustre de Leon, J., Zabinsky, S.I. & Albers, R.C. Theoretical x-ray absorption fine standards. J. Am. Chem. Soc. 113, 5135–5140 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank N. Echols and T. Alber for their help in the X-ray data collection for the selenomethionine-CsoR crystals, and we thank X. Chen for many helpful discussions. This work was supported by grants from the US National Institutes of Health (GM042569 to D.P.G. and AI068135 to J.C.S.), the Robert A. Welch Foundation (A1295 to D.P.G.) and the US Department of Agriculture (WIS04794 and WIS04823 to A.M.T.). S.K.W. was supported by a US National Institutes of Health Molecular Biosciences Training Grant (T32 GM007215). SSRL is funded by the US Department of Energy and the US National Institutes of Health. The CLS is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the National Research Council Canada, the Canadian Institutes of Health Research (CIHR) and the University of Saskatchewan. Research at the University of Saskatchewan was supported by the NSERC and the CIHR, a Canada Research Chair award (G.N.G.), and the University of Saskatchewan, the Province of Saskatchewan, and the US National Institutes of Health (GM057375).

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Author notes

  1. Tong Liu and Arati Ramesh: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, 77843-2128, USA

    Tong Liu, Arati Ramesh, Zhen Ma, James C Sacchettini & David P Giedroc

  2. Center for Structural Biology, Texas A&M University, College Station, Texas, 77843-2128, USA

    Arati Ramesh & James C Sacchettini

  3. Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Sarah K Ward & Adel M Talaat

  4. Department of Geological Sciences, University of Saskatchewan, Saskatoon, S7N 5E2, Saskatchewan, Canada

    Limei Zhang & Graham N George

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Contributions

T.L. designed and performed biochemical experiments; A.R. solved the crystallographic structure of CsoR; Z.M. performed metal and DNA binding experiments with mutant CsoRs; S.K.W. performed metal inducibility experiments in Mtb cultures; L.Z. performed XAS experiments on samples prepared by T.L.; G.N.G. analyzed the XAS data and edited the manuscript; A.D.T. analyzed Mtb experiments; J.C.S. analyzed the crystallographic experiments and edited the manuscript; D.P.G. designed the experimental plan and wrote the manuscript.

Corresponding authors

Correspondence to James C Sacchettini or David P Giedroc.

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Supplementary information

Supplementary Fig. 1

The rv0967–rv0970 operon is expressed as a polycistronic operon in Mtb. (PDF 1203 kb)

Supplementary Fig. 2

The expression from the cso promoter is specifically induced by addition of copper salts to the growth medium in M. smegmatis. (PDF 361 kb)

Supplementary Fig. 3

Mapping the CsoR-DNA binding fragment in the operator/promoter region of cso operon by EMSA. (PDF 3336 kb)

Supplementary Fig. 4

Two distinct forms of recombinant CsoR can be purified from E. coli using nondenaturing methods and SDS-PAGE analysis of various Mtb CsoR samples. (PDF 7097 kb)

Supplementary Fig. 5

Copper and DNA binding properties of variant apo-CsoRs. (PDF 836 kb)

Supplementary Table 1

Crystallographic data collection and refinement statistics for Mtb CsoR. (PDF 75 kb)

Supplementary Table 2

EXAFS curve-fitting results. (PDF 75 kb)

Supplementary Methods

Additional details on methods used and physical characterization of CsoR. (PDF 248 kb)

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Liu, T., Ramesh, A., Ma, Z. et al. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator. Nat Chem Biol 3, 60–68 (2007). https://doi.org/10.1038/nchembio844

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