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.

Bacterial sensors define intracellular free energies for correct enzyme metalation

Nature Chemical Biology volume 15, pages 241–249 (2019)Cite this article

Subjects

Abstract

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Fig. 1: Metal binding and DNA binding are coupled to enable Salmonella to sense different metals.
Fig. 2: Metal affinities that complete a set of values for Salmonella metal sensors.
Fig. 3: Metals change the abundance of some sensors to modify regulation.
Fig. 4: Sensing is tuned to the Irving–Williams series.
Fig. 5: Metalation of CbiK and sirohydrochlorin.

Code availability

Equation derivations, template Excel spreadsheet (with instructions) and MATLAB codes (with instructions) are available in Supplementary Note 2, Supplementary Dataset 1 and Supplementary Note 3, respectively.

Data availability

All data are available within the article and its Supplementary Information files or from the corresponding author upon request.

References

  1. Waldron, K. J., Rutherford, J. C., Ford, D. & Robinson, N. J. Metalloproteins and metal sensing. Nature 460, 823–830 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Tottey, S. et al. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 455, 1138–1142 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Fraústo da Silva, J. J. R. & Williams, R. J. P. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life (Oxford Univ. Press, Oxford, 1991).

    Google Scholar 

  4. Irving, H. & Williams, R. J. P. Order of stability of metal complexes. Nature 162, 746–747 (1948).

    Article  CAS  Google Scholar 

  5. Chandrangsu, P., Rensing, C. & Helmann, J. D. Metal homeostasis and resistance in bacteria. Nat. Rev. Microbiol. 15, 338–350 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Reyes-Caballero, H., Campanello, G. C. & Giedroc, D. P. Metalloregulatory proteins: metal selectivity and allosteric switching. Biophys. Chem. 156, 103–114 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Giedroc, D. P. & Arunkumar, A. I. Metal sensor proteins: nature’s metalloregulated allosteric switches. Dalton. Trans. 29, 3107–3120 (2007).

    Article  Google Scholar 

  8. Foster, A. W. et al. A tight tunable range for Ni(ii) sensing and buffering in cells. Nat. Chem. Biol. 13, 409–414 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Outten, C. E. & O’Halloran, T. V. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292, 2488–2492 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Carter, K. P., Young, A. M. & Palmer, A. E. Fluorescent sensors for measuring metal ions in living systems. Chem. Rev. 114, 4564–4601 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Osman, D. et al. Fine control of metal concentrations is necessary for cells to discern zinc from cobalt. Nat. Commun. 8, 1884 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Ansari, A. Z., Chael, M. L. & O’Halloran, T. V. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87–89 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Ikeda, J. S., Janakiraman, A., Kehres, D. G., Maguire, M. E. & Slauch, J. M. Transcriptional regulation of sitABCD of Salmonella enterica serovar Typhimurium by MntR and Fur. J. Bacteriol. 187, 912–922 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. O’Halloran, T. & Walsh, C. Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization. Science 235, 211–214 (1987).

    Article  PubMed  Google Scholar 

  16. 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  PubMed  Google Scholar 

  17. Iwig, J. S., Leitch, S., Herbst, R. W., Maroney, M. J. & Chivers, P. T. Ni(ii) and Co(ii) sensing by Escherichia coli RcnR. J. Am. Chem. Soc. 130, 7592–7606 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Que, Q. & Helmann, J. D. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol. Microbiol. 35, 1454–1468 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Althaus, E. W., Outten, C. E., Olson, K. E., Cao, H. & O’Halloran, T. V. The ferric uptake regulation (Fur) repressor is a zinc metalloprotein. Biochemistry 38, 6559–6569 (1999).

    Article  CAS  PubMed  Google Scholar 

  20. Hantke, K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol. Gen. Genet. 182, 288–292 (1981).

    Article  CAS  PubMed  Google Scholar 

  21. Patzer, S. I. & Hantke, K. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli. Mol. Microbiol. 28, 1199–1210 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Schreiter, E. R. et al. Crystal structure of the nickel-responsive transcription factor NikR. Nat. Struct. Biol. 10, 794–799 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Osman, D. et al. Generating a metal-responsive transcriptional regulator to test what confers metal sensing in cells. J. Biol. Chem. 290, 19806–19822 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pi, H. & Helmann, J. D. Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis. Proc. Natl Acad. Sci. USA 114, 12785–12790 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Record, M. T. Jr., Ha, J. H. & Fisher, M. A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA. Methods Enzymol. 208, 291–343 (1991).

    Article  CAS  PubMed  Google Scholar 

  26. Campanello, G. C. et al. Allosteric inhibition of a zinc-sensing transcriptional repressor: insights into the arsenic repressor (ArsR) family. J. Mol. Biol. 425, 1143–1157 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Epstein, W. & Schultz, S. G. Cation Transport in Escherichia coli: V. Regulation of cation content. J. Gen. Physiol. 49, 221–234 (1965).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Su, J., Gong, H., Lai, J., Main, A. & Lu, S. The potassium transporter Trk and external potassium modulate Salmonella enterica protein secretion and virulence. Infect. Immun. 77, 667–675 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Stickle, D. F., Vossen, K. M., Riley, D. A. & Fried, M. G. Free DNA concentration in E. coli estimated by an analysis of competition for DNA binding proteins. J. Theor. Biol. 168, 1–12 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Gilston, B. A. et al. Structural and mechanistic basis of zinc regulation across the E. coli Zur regulon. PLoS Biol. 12, e1001987 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Shin, J.-H. & Helmann, J. D. Molecular logic of the Zur-regulated zinc deprivation response in Bacillus subtilis. Nat. Commun. 7, 12612 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ma, Z., Gabriel, S. E. & Helmann, J. D. Sequential binding and sensing of Zn(ii) by Bacillus subtilis Zur. Nucleic Acids Res. 39, 9130–9138 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Natori, Y. et al. A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis. Mol. Microbiol. 63, 294–307 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Dalecki, A. G., Crawford, C. L. & Wolschendorf, F. Copper and antibiotics: discovery, modes of action, and opportunities for medicinal applications. Adv. Microb. Physiol. 70, 193–260 (2017).

    Article  PubMed  Google Scholar 

  35. Foster, A. W., Osman, D. & Robinson, N. J. Metal preferences and metallation. J. Biol. Chem. 289, 28095–28103 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Warren, M. J., Raux, E., Schubert, H. L. & Escalante-Semerena, J. C. The biosynthesis of adenosylcobalamin (vitamin B12). Nat. Prod. Rep. 19, 390–412 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Frank, S. et al. Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway. Biochem. Soc. Trans. 33, 811–814 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Raux, E., Thermes, C., Heathcote, P., Rambach, A. & Warren, M. J. A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis. J. Bacteriol. 179, 3202–3212 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lobo, S. A. et al. Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris hildenborough. Biochemistry 47, 5851–5857 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Martin, J. E., Lisher, J. P., Winkler, M. E. & Giedroc, D. P. Perturbation of manganese metabolism disrupts cell division in Streptococcus pneumoniae. Mol. Microbiol. 104, 334–348 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Brown, D. R. et al. The cellular prion protein binds copper in vivo. Nature 390, 684–687 (1997).

    Article  CAS  Google Scholar 

  42. Bush, A. I. The metallobiology of Alzheimer’s disease. Trends Neurosci. 26, 207–214 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. McCarthy, R. C. & Kosman, D. J. Iron transport across the blood-brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy. Cell. Mol. Life Sci. 72, 709–727 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Xiao, T. et al. Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system. Nat. Chem. Biol. 14, 655–663 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hood, M. I. & Skaar, E. P. Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Microbiol. 10, 525–537 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Lisher, J. P. & Giedroc, D. P. Manganese acquisition and homeostasis at the host-pathogen interface. Front. Cell. Infect. Microbiol. 3, 91 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Wilks, A. & Burkhard, K. A. Heme and virulence: how bacterial pathogens regulate, transport and utilize heme. Nat. Prod. Rep. 24, 511–522 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Djoko, K. Y., Ong, C. L., Walker, M. J. & McEwan, A. G. The role of copper and zinc toxicity in innate immune defense against bacterial pathogens. J. Biol. Chem. 290, 18954–18961 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Damo, S. M. et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc. Natl. Acad. Sci. USA 110, 3841–3846 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Lemire, J. A., Harrison, J. J. & Turner, R. J. Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat. Rev. Microbiol. 11, 371–384 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Chivers, P. T., Benanti, E. L., Heil-Chapdelaine, V., Iwig, J. S. & Rowe, J. L. Identification of Ni-(l-His)2 as a substrate for NikABCDE-dependent nickel uptake in Escherichia coli. Metallomics 4, 1043–1050 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Δ Δ C(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

  53. Ramakers, C., Ruijter, J. M., Deprez, R. H. L. & Moorman, A. F. M. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62–66 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Chivers, P. T. & Sauer, R. T. NikR repressor: high-affinity nickel binding to the C-terminal domain regulates binding to operator DNA. Chem. Biol. 9, 1141–1148 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Romão, C. V. et al. Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization. Proc. Natl. Acad. Sci. USA 108, 97–102 (2011).

    Article  PubMed  Google Scholar 

  56. Stookey, L. L. Ferrozine-a new spectrophotometric reagent for iron. Anal. Chem. 42, 779–781 (1970).

    Article  CAS  Google Scholar 

  57. Dainty, S. J., Patterson, C. J., Waldron, K. J. & Robinson, N. J. Interaction between cyanobacterial copper chaperone Atx1 and zinc homeostasis. J. Biol. Inorg. Chem. 15, 77–85 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  59. Reyes-Caballero, H., Lee, C. W. & Giedroc, D. P. Mycobacterium tuberculosis NmtR harbors a nickel sensing site with parallels to Escherichia coli RcnR. Biochemistry 50, 7941–7952 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by Biotechnology and Biological Sciences Research Council awards nos. BB/J017787/1, BB/R002118/1 and BB/L009226/1. Interactions with industrial partners were supported by Biotechnology and Biological Sciences Research Council (BBSRC) award no. BB/L013711/1 plus a financial contribution from Procter and Gamble (in association with an Industrial Partnership Award no. BB/J017787/1). K. Svedaite, Department of Biosciences, Durham University, provided technical assistance in the measurements of in vitro DNA affinities of ZntR and CueR. E. Pohl and C. Bain, both of Durham University Department of Chemistry, assisted with structure homology modeling and consideration of standard free-energy changes, respectively. Salmonella enterica serovar Typhimurium strain SL1344 was provided by J.S. Cavet, School of Biological Sciences, University of Manchester, Manchester, UK. E. Fioravanti, Mathematical Institute, University of Oxford, assisted with derivations shown in the Supplementary Note 2. All DNA sequencing was conducted by DBS genomics, Durham University.

Author information

Author notes

  1. These authors contributed equally: Deenah Osman, Maria Alessandra Martini, Andrew W. Foster.

Authors and Affiliations

  1. Department of Biosciences, Durham University, Durham, UK

    Deenah Osman, Maria Alessandra Martini, Andrew W. Foster, Andrew J. P. Scott, Elena Lurie-Luke, Peter T. Chivers & Nigel J. Robinson

  2. Department of Chemistry, Durham University, Durham, UK

    Deenah Osman, Andrew W. Foster, Jonathan W. Steed, Peter T. Chivers & Nigel J. Robinson

  3. Procter and Gamble, Mason Business Center, Cincinnati, OH, USA

    Junjun Chen & Thomas G. Huggins

  4. Department of Mathematics, Physics and Electrical Engineering, Northumbria University, Newcastle-upon-Tyne, UK

    Richard J. Morton

  5. School of Biosciences, University of Kent, Canterbury, Kent, UK

    Andrew D. Lawrence, Evelyne Deery & Martin J. Warren

Authors
  1. Deenah Osman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Alessandra Martini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew W. Foster
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Junjun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew J. P. Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard J. Morton
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jonathan W. Steed
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Elena Lurie-Luke
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thomas G. Huggins
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Andrew D. Lawrence
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Evelyne Deery
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Martin J. Warren
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Peter T. Chivers
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Nigel J. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.O. conducted the in vivo experiments, bioinformatics analyses and was involved in all in vitro measurements of sensor affinities. M.A.M. determined in vitro affinities of MntR and Fur. M.A.M., along with D.O., A.W.F. and J.W.S., developed computational methods to determine θD and θDM. R.J.M. along with D.O. generated the MATLAB code relating fractional sensor responses to buffered [M]. A.J.P.S. and P.T.C. determined the in vitro affinities of NikR. J.C. and T.G.H. performed the MRM tandem mass spectrometry. A.W.F. along with E.D., A.D.L., P.T.C. and M.J.W. performed and co-designed analyses of CbiK. N.J.R. and E.L.-L. conceived the program. N.J.R., D.O. and A.W.F. drafted the manuscript and, in conjunction with M.A.M., interpreted the significance of the data. N.J.R., with input from P.T.C., had overall responsibility for the design, coordination and management of the project. All authors reviewed the results and edited and approved the final version of the manuscript.

Corresponding authors

Correspondence to Peter T. Chivers or Nigel J. Robinson.

Ethics declarations

Competing interests

J.C. and T.G.H. are employees of Procter and Gamble. The collaboration was supported by an Industrial Partnership Award from the BBSRC plus a financial contribution from Procter and Gamble (in association with BBSRC award no. BB/J017787/1).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–5, Supplementary Figures 1–22

Reporting Summary

Supplementary Dataset 1

Excel Spreadsheet (with instructions) to enable calculation of fractional DNA occupancy

Supplementary Note 1

The Dynafit scripts

Supplementary Note 2

The supplementary equations and unique Supplementary Note 2 references

Supplementary Note 3

The MATLAB codes (with instructions), to determine the buffered metal concentration from given value(s) of θD or θDM

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Osman, D., Martini, M.A., Foster, A.W. et al. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat Chem Biol 15, 241–249 (2019). https://doi.org/10.1038/s41589-018-0211-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0211-4

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