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:

A new structural paradigm in copper resistance in Streptococcus pneumoniae

Nature Chemical Biology volume 9, pages 177–183 (2013)Cite this article

Subjects

Abstract

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA and the copper-effluxing P1B-type ATPase CopA. We show here that CupA is a previously uncharacterized cell membrane–anchored Cu(I) chaperone and that a Cu(I) binding–competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA and the N-terminal metal-binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds that each harbor a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on the soluble domain of CupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

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: Copper sensitivity phenotypes of mutant S. pneumoniae D39 strains.
Figure 2: Crystallographic structures of sCupA and CopAMBD.
Figure 3: The methionine-rich S2 site is the low-affinity site on both CopAMBD and sCupA, and Cu(I) is transferred only from the S2 site of sCupA to the S1 site of apo MBD.
Figure 4: NMR chemical shift perturbation analysis of sCupA and CopAMBD induced by Cu(I) binding.
Figure 5: Mutagenesis of Cu(I)-binding residues in CupA but not in CopAMBD partly or completely abrogates Cu(I) resistance of S. pneumoniae.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Peña, M.M., Lee, J. & Thiele, D.J. A delicate balance: homeostatic control of copper uptake and distribution. J. Nutr. 129, 1251–1260 (1999).

    PubMed  Google Scholar 

  2. Halliwell, B. & Gutteridge, J.M. The importance of free radicals and catalytic metal ions in human diseases. Mol. Aspects Med. 8, 89–193 (1985).

    CAS  PubMed  Google Scholar 

  3. Percival, S.S. Copper and immunity. Am. J. Clin. Nutr. 67, 1064S–1068S (1998).

    CAS  PubMed  Google Scholar 

  4. Braun, V. & Hantke, K. Recent insights into iron import by bacteria. Curr. Opin. Chem. Biol. 15, 328–334 (2011).

    CAS  PubMed  Google Scholar 

  5. Corbin, B.D. et al. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science 319, 962–965 (2008).

    CAS  PubMed  Google Scholar 

  6. Samanovic, M.I., Ding, C., Thiele, D.J. & Darwin, K.H. Copper in microbial pathogenesis: meddling with the metal. Cell Host Microbe 11, 106–115 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Macomber, L. & Imlay, J.A. The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc. Natl. Acad. Sci. USA 106, 8344–8349 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Tottey, S. et al. Cyanobacterial metallochaperone inhibits deleterious side reactions of copper. Proc. Natl. Acad. Sci. USA 109, 95–100 (2012).

    CAS  PubMed  Google Scholar 

  9. Baker, J. et al. Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation. Appl. Environ. Microbiol. 76, 150–160 (2010).

    CAS  PubMed  Google Scholar 

  10. Ward, S.K., Hoye, E.A. & Talaat, A.M. The global responses of Mycobacterium tuberculosis to physiological levels of copper. J. Bacteriol. 190, 2939–2946 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. White, C., Lee, J., Kambe, T., Fritsche, K. & Petris, M.J. A role for the ATP7A copper-transporting ATPase in macrophage bactericidal activity. J. Biol. Chem. 284, 33949–33956 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Osman, D. et al. Copper homeostasis in Salmonella is atypical and copper-CueP is a major periplasmic metal complex. J. Biol. Chem. 285, 25259–25268 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gold, B. et al. Identification of a copper-binding metallothionein in pathogenic mycobacteria. Nat. Chem. Biol. 4, 609–616 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Festa, R.A. et al. A novel copper-responsive regulon in Mycobacterium tuberculosis. Mol. Microbiol. 79, 133–148 (2011).

    CAS  PubMed  Google Scholar 

  15. Wolschendorf, F. et al. Copper resistance is essential for virulence of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 108, 1621–1626 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Rowland, J.L. & Niederweis, M. Resistance mechanisms of Mycobacterium tuberculosis against phagosomal copper overload. Tuberculosis (Edinb.) 92, 202–210 (2012).

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  18. Shafeeq, S. et al. The cop operon is required for copper homeostasis and contributes to virulence in Streptococcus pneumoniae. Mol. Microbiol. 81, 1255–1270 (2011).

    CAS  PubMed  Google Scholar 

  19. Kazmierczak, K.M., Wayne, K.J., Rechtsteiner, A. & Winkler, M.E. Roles of rel(Spn) in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39. Mol. Microbiol. 72, 590–611 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Jacobsen, F.E., Kazmierczak, K.M., Lisher, J.P., Winkler, M.E. & Giedroc, D.P. Interplay between manganese and zinc homeostasis in the human pathogen Streptococcus pneumoniae. Metallomics 3, 38–41 (2011).

    CAS  PubMed  Google Scholar 

  21. Hava, D.L. & Camilli, A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol. Microbiol. 45, 1389–1406 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Corbett, D. et al. The combined actions of the copper-responsive repressor CsoR and copper-metallochaperone CopZ modulate CopA-mediated copper efflux in the intracellular pathogen Listeria monocytogenes. Mol. Microbiol. 81, 457–472 (2011).

    CAS  PubMed  Google Scholar 

  23. Badarau, A. & Dennison, C. Copper trafficking mechanism of CXXC-containing domains: insight from the pH-dependence of their Cu(I) affinities. J. Am. Chem. Soc. 133, 2983–2988 (2011).

    CAS  PubMed  Google Scholar 

  24. Pufahl, R.A. et al. Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science 278, 853–856 (1997).

    CAS  PubMed  Google Scholar 

  25. Arnesano, F. et al. Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase. J. Biol. Chem. 276, 41365–41376 (2001).

    CAS  PubMed  Google Scholar 

  26. Banci, L. et al. A NMR study of the interaction of a three-domain construct of ATP7A with copper(I) and copper(I)-HAH1: the interplay of domains. J. Biol. Chem. 280, 38259–38263 (2005).

    CAS  PubMed  Google Scholar 

  27. Banci, L. et al. The delivery of copper for thylakoid import observed by NMR. Proc. Natl. Acad. Sci. USA 103, 8320–8325 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Banci, L., Bertini, I., Del Conte, R., Markey, J. & Ruiz-Duenas, F.J. Copper trafficking: the solution structure of Bacillus subtilis CopZ. Biochemistry 40, 15660–15668 (2001).

    CAS  PubMed  Google Scholar 

  29. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Banci, L. et al. A copper(I) protein possibly involved in the assembly of CuA center of bacterial cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 102, 3994–3999 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Abriata, L.A. et al. Mechanism of Cu(A) assembly. Nat. Chem. Biol. 4, 599–601 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Gourdon, P. et al. Crystal structure of a copper-transporting PIB-type ATPase. Nature 475, 59–64 (2011).

    CAS  PubMed  Google Scholar 

  33. Rosenzweig, A.C. Copper delivery by metallochaperone proteins. Acc. Chem. Res. 34, 119–128 (2001).

    CAS  PubMed  Google Scholar 

  34. Xiao, Z. et al. Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards. J. Biol. Chem. 286, 11047–11055 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Dupont, C.L., Grass, G. & Rensing, C. Copper toxicity and the origin of bacterial resistance—new insights and applications. Metallomics 3, 1109–1118 (2011).

    CAS  PubMed  Google Scholar 

  36. Rosenzweig, A.C. Metallochaperones: bind and deliver. Chem. Biol. 9, 673–677 (2002).

    CAS  PubMed  Google Scholar 

  37. Banci, L. et al. Affinity gradients drive copper to cellular destinations. Nature 465, 645–648 (2010).

    CAS  PubMed  Google Scholar 

  38. Badarau, A. & Dennison, C. Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803. Proc. Natl. Acad. Sci. USA 108, 13007–13012 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zaballa, M.E., Abriata, L.A., Donaire, A. & Vila, A.J. Flexibility of the metal-binding region in apo-cupredoxins. Proc. Natl. Acad. Sci. USA 109, 9254–9259 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Bersch, B. et al. Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34. Biochemistry 50, 2194–2204 (2011).

    CAS  PubMed  Google Scholar 

  41. Wu, C.C., Rice, W.J. & Stokes, D.L. Structure of a copper pump suggests a regulatory role for its metal-binding domain. Structure 16, 976–985 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. González-Guerrero, M. & Arguello, J.M. Mechanism of Cu+-transporting ATPases: soluble Cu+ chaperones directly transfer Cu+ to transmembrane transport sites. Proc. Natl. Acad. Sci. USA 105, 5992–5997 (2008).

    PubMed  PubMed Central  Google Scholar 

  43. Chillappagari, S., Miethke, M., Trip, H., Kuipers, O.P. & Marahiel, M.A. Copper acquisition is mediated by YcnJ and regulated by YcnK and CsoR in Bacillus subtilis. J. Bacteriol. 191, 2362–2370 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Ekici, S., Yang, H., Koch, H.G. & Daldal, F. Novel transporter required for biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus. MBio. 3, e00293–e00311 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ramos-Montañez, S., Kazmierczak, K.M., Hentchel, K.L. & Winkler, M.E. Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39. J. Bacteriol. 192, 6390–6400 (2010).

    PubMed  PubMed Central  Google Scholar 

  46. Anjem, A. & Imlay, J.A. Mononuclear iron enzymes are primary targets of hydrogen peroxide stress. J. Biol. Chem. 287, 15544–15556 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Lynch, J.P. III & Zhanel, G.G. Streptococcus pneumoniae: epidemiology and risk factors, evolution of antimicrobial resistance, and impact of vaccines. Curr. Opin. Pulm. Med. 16, 217–225 (2010).

    PubMed  Google Scholar 

  48. Lanie, J.A. et al. Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J. Bacteriol. 189, 38–51 (2007).

    CAS  PubMed  Google Scholar 

  49. Ramos-Montañez, S. et al. Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae. Mol. Microbiol. 67, 729–746 (2008).

    PubMed  Google Scholar 

  50. Wayne, K.J. et al. Localization and cellular amounts of the WalRKJ (VicRKX) two-component regulatory system proteins in serotype 2 Streptococcus pneumoniae. J. Bacteriol. 192, 4388–4394 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kuzmič, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260–273 (1996).

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  54. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Google Scholar 

  55. Lescop, E., Rasia, R. & Brutscher, B. Hadamard amino-acid-type edited NMR experiment for fast protein resonance assignment. J. Am. Chem. Soc. 130, 5014–5015 (2008).

    CAS  PubMed  Google Scholar 

  56. Lescop, E., Schanda, P. & Brutscher, B. A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J. Magn. Reson. 187, 163–169 (2007).

    CAS  PubMed  Google Scholar 

  57. Webb, S.M. SIXpack: a graphical user interface for XAS analysis using IFEFFIT. Phys. Scr. 115, 1011–1014 (2005).

    Google Scholar 

  58. Zabinsky, S.I., Rehr, J.J., Ankudinov, A., Albers, R.C. & Eller, M.J. Multiple-scattering calculations of X-ray-absorption spectra. Phys. Rev. B Condens. Matter 52, 2995–3009 (1995).

    CAS  PubMed  Google Scholar 

  59. Ankudinov, A.L., Ravel, B., Rehr, J.J. & Conradson, S.D. Real-space multiple-scattering calculation and interpretation of X-ray-absorption near-edge structure. Phys. Rev. B 58, 7565–7576 (1998).

    CAS  Google Scholar 

  60. Leitch, S., Bradley, M.J., Rowe, J.L., Chivers, P.T. & Maroney, M.J. Nickel-specific response in the transcriptional regulator, Escherichia coli NikR. J. Am. Chem. Soc. 129, 5085–5095 (2007).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge support from the US National Institutes of Health (NIH; GM042569 to D.P.G., GM094472 to C.E.D. III, AI095814 to M.E.W. and GM069696 to M.J.M.), the Lilly Endowment (to M.E.W.) and an Indiana University Quantitative and Chemical Biology Training Fellowship (to J.P.L.). We thank L. Christiansen for assistance in strain construction, members of the Giedroc laboratory for help in acquiring the NMR data and H. Hu and N. Giri from of the Maroney laboratory for XAS data collection. Crystallographic data collection at the Advanced Light Source at the Lawrence Berkeley National Laboratory is supported by the US Department of Energy and Indiana University. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research and by the NIH National Center for Research Resources, Biomedical Technology Program. XAS data collected at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory was supported by the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. Beamline X3B at NSLS is supported by the Center for Synchrotron Biosciences (grant P30-EB-009998) from the National Institute of Biomedical Imaging and Bioengineering.

Author information

Authors and Affiliations

  1. Department of Chemistry, Indiana University, Bloomington, Indiana, USA

    Yue Fu, Khadine A Higgins, John P Lisher, Charles E Dann III & David P Giedroc

  2. Interdisciplinary Graduate Program in Biochemistry, Indiana University, Bloomington, Indiana, USA

    Yue Fu, John P Lisher, Charles E Dann III, Malcolm E Winkler & David P Giedroc

  3. Department of Biology, Indiana University, Bloomington, Indiana, USA

    Ho-Ching Tiffany Tsui, Kevin E Bruce, Lok-To Sham, Krystyna M Kazmierczak & Malcolm E Winkler

  4. Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA

    Michael J Maroney

Authors
  1. Yue Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ho-Ching Tiffany Tsui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kevin E Bruce
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lok-To Sham
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Khadine A Higgins
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John P Lisher
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Krystyna M Kazmierczak
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael J Maroney
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Charles E Dann III
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Malcolm E Winkler
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. David P Giedroc
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.F. carried out all protein purification, Cu(I) binding experiments and NMR studies, and solved the crystallographic structures of sCupA and CopAMBD, the latter in collaboration with C.E.D. III., H.-C.T.T., K.E.B. and L.-T.S. K.M.K. constructed S. pneumoniae strains and carried out all cell culture and western blotting experiments under the direction of M.E.W. K.A.H. prepared samples for XAS and analyzed these spectra in collaboration with M.J.M., and J.P.L. made the ICP-MS measurements on cultures grown by K.E.B. D.P.G. conceived and directed the study, and wrote the manuscript.

Corresponding author

Correspondence to David P Giedroc.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results (PDF 7636 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fu, Y., Tsui, HC., Bruce, K. et al. A new structural paradigm in copper resistance in Streptococcus pneumoniae. Nat Chem Biol 9, 177–183 (2013). https://doi.org/10.1038/nchembio.1168

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1168

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