Experimental phasing using zinc and sulfur anomalous signals measured at the zinc absorption peak

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

An Erratum to this article was published

Abstract

Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization, and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.

This is a preview of subscription content, log in to check access.

References

  1. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., and et al. 2010. Phenix: A comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221.

    PubMed  Article  CAS  Google Scholar 

  2. Berg, J.M. and Shi, Y. 1996. The galvanization of biology: A growing appreciation for the roles of zinc. Science 271, 1081–1085.

    PubMed  Article  CAS  Google Scholar 

  3. Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., and et al. 1998. Crystallography & NMR System: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.

    PubMed  Article  Google Scholar 

  4. Butcher, J., Sarvan, S., Brunzelle, J.S., Couture, J.F., and Stintzi, A. 2012. Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation. Proc. Natl. Acad. Sci. USA 109, 10047–10052.

    PubMed  Article  CAS  Google Scholar 

  5. Cha, S.S., An, Y.J., Jeong, C.S., Kim, M.K., Lee, S.G., Lee, K.H., and Oh, B.H. 2012. Experimental phasing using zinc anomalous scattering. Acta Crystallogr. D Biol. Crystallogr. 68, 1253–1258.

    PubMed  Article  CAS  Google Scholar 

  6. da Silva Neto, J.F., Braz, V.S., Italiani, V.C., and Marques, M.V. 2009. Fur controls iron homeostasis and oxidative stress defense in the oligotrophic α-Proteobacterium Caulobacter crescentus. Nucleic Acids Res. 37, 4812–4825.

    PubMed  Article  Google Scholar 

  7. Delany, I., Pacheco, A.B., Spohn, G., Rappuoli, R., and Scarlato, V. 2001a. Iron-dependent transcription of the frpb gene of Helicobacter pylori is controlled by the Fur repressor protein. J. Bacteriol. 183, 4932–4937.

    PubMed  Article  CAS  Google Scholar 

  8. Delany, I., Spohn, G., Rappuoli, R., and Scarlato, V. 2001b. The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol. Microbiol. 42, 1297–1309.

    PubMed  Article  CAS  Google Scholar 

  9. Dian, C., Vitale, S., Leonard, G.A., Bahlawane, C., Fauquant, C., Leduc, D., Muller, C., de Reuse, H., Michaud-Soret, I., and Terradot, L. 2011. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites. Mol. Microbiol. 79, 1260–1275.

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  Google Scholar 

  11. Escolar, L., Perez-Martin, J., and de Lorenzo, V. 1999. Opening the iron box: Transcriptional metalloregulation by the Fur protein. J. Bacteriol. 181, 6223–6229.

    PubMed  CAS  Google Scholar 

  12. Gancz, H., Censini, S., and Merrell, D.S. 2006. Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori. Infect. Immun. 74, 602–614.

    PubMed  Article  CAS  Google Scholar 

  13. Grosse-Kunstleve, R.W. and Adams, P.D. 2003. Substructure search procedures for macromolecular structures. Acta Crystallogr. D Biol. Crystallogr. 59, 1966–1973.

    PubMed  Article  CAS  Google Scholar 

  14. Haber, F. and Weiss, J. 1932. On the catalysis of dydroperoxide. Naturwissenschaften 20, 948–950.

    Article  CAS  Google Scholar 

  15. Hernandez, J.A., Bes, M.T., Fillat, M.F., Neira, J.L., and Peleato, M.L. 2002. Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state. Biochem. J. 366, 315–322.

    PubMed  CAS  Google Scholar 

  16. Kadner, R.J. 2005. Regulation by iron: RNA rules the rust. J. Bacteriol. 187, 6870–6873.

    PubMed  Article  CAS  Google Scholar 

  17. Kim, M.K., Lee, S., An, Y.J., Jeong, C.S., Ji, C.J., Lee, J.W., and Cha, S.S. 2013. In-house zinc SAD phasing at Cu kα edge. Mol. Cells 36, 74–81.

    PubMed  Article  CAS  Google Scholar 

  18. Lee, H.J., Bang, S.H., Lee, K.H., and Park, S.J. 2007. Positive regulation of fur gene expression via direct interaction of Fur in a pathogenic bacterium, Vibrio vulnificus. J. Bacteriol. 189, 2629–2636.

    PubMed  Article  CAS  Google Scholar 

  19. Lee, J.W. and Helmann, J.D. 2006. Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR. J. Biol. Chem. 281, 23567–23578.

    PubMed  Article  CAS  Google Scholar 

  20. Lee, J.W. and Helmann, J.D. 2007. Functional specialization within the Fur family of metalloregulators. Biometals 20, 485–499.

    PubMed  Article  CAS  Google Scholar 

  21. Ma, Z., Faulkner, M.J., and Helmann, J.D. 2012. Origins of specificity and cross-talk in metal ion sensing by Bacillus subtilis Fur. Mol. Microbiol. 86, 1144–1155.

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  23. McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. 2007. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674.

    PubMed  Article  CAS  Google Scholar 

  24. McHugh, J.P., Rodriguez-Quinones, F., Abdul-Tehrani, H., Svistunenko, D.A., Poole, R.K., Cooper, C.E., and Andrews, S.C. 2003. Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J. Biol. Chem. 278, 29478–29486.

    PubMed  Article  CAS  Google Scholar 

  25. Nicholls, R.A., Long, F., and Murshudov, G.N. 2012. Low-resolution refinement tools in 5 Acta Crystallogr. D Biol. Crystallogr. 68, 404–417.

    Article  CAS  Google Scholar 

  26. Otwinowski, Z. and Minor, W. 1997. Processing of x-ray diffraction data collected in oscillation mode. Method Enzymol. 276, 307–326.

    Article  CAS  Google Scholar 

  27. Palyada, K., Threadgill, D., and Stintzi, A. 2004. Iron acquisition and regulation in Campylobacter jejuni. J. Bacteriol. 186, 4714–4729.

    PubMed  Article  CAS  Google Scholar 

  28. Pohl, E., Haller, J.C., Mijovilovich, A., Meyer-Klaucke, W., Garman, E., and Vasil, M.L. 2003. Architecture of a protein central to iron homeostasis: Crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol. Microbiol. 47, 903–915.

    PubMed  Article  CAS  Google Scholar 

  29. Ramagopal, U.A., Dauter, M., and Dauter, Z. 2003. Phasing on anomalous signal of sulfurs: What is the limit? Acta Crystallogr. D Biol. Crystallogr. 59, 1020–1027.

    PubMed  Article  Google Scholar 

  30. Schaible, U.E. and Kaufmann, S.H. 2004. Iron and microbial infection. Nat. Rev. Microbiol. 2, 946–953.

    PubMed  Article  CAS  Google Scholar 

  31. Sheikh, M.A. and Taylor, G.L. 2009. Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination. Mol. Microbiol. 72, 1208–1220.

    PubMed  Article  CAS  Google Scholar 

  32. Sheldrick, G.M. 2010. Experimental phasing with shelxc/d/e: Combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485.

    PubMed  Article  CAS  Google Scholar 

  33. Shin, J.H., Jung, H.J., An, Y.J., Cho, Y.B., Cha, S.S., and Roe, J.H. 2011. Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur. Proc. Natl. Acad. Sci. USA 108, 5045–5050.

    PubMed  Article  CAS  Google Scholar 

  34. Terwilliger, T.C. 2002. Automated structure solution, density modification and model building. Acta Crystallogr. D Biol. Crystallogr. 58, 1937–1940.

    PubMed  Article  Google Scholar 

  35. Terwilliger, T.C. 2003. Solve and resolve: Automated structure solution and density modification. Methods Enzymol. 374, 22–37.

    PubMed  Article  CAS  Google Scholar 

  36. Terwilliger, T.C., Adams, P.D., Read, R.J., McCoy, A.J., Moriarty, N.W., Grosse-Kunstleve, R.W., Afonine, P.V., Zwart, P.H., and Hung, L.W. 2009. Decision-making in structure solution using Bayesian estimates of map quality: The PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582–601.

    PubMed  Article  CAS  Google Scholar 

  37. Torres, V.J., Attia, A.S., Mason, W.J., Hood, M.I., Corbin, B.D., Beasley, F.C., Anderson, K.L., Stauff, D.L., McDonald, W.H., Zimmerman, L.J., and et al. 2010. Staphylococcus aureus fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia. Infect. Immun. 78, 1618–1628.

    PubMed  Article  CAS  Google Scholar 

  38. Traore, D.A., El Ghazouani, A., Ilango, S., Dupuy, J., Jacquamet, L., Ferrer, J.L., Caux-Thang, C., Duarte, V., and Latour, J.M. 2006. Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis. Mol. Microbiol. 61, 1211–1219.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Jin-Won Lee or Sun-Shin Cha.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, S., Kim, M., Ji, C. et al. Experimental phasing using zinc and sulfur anomalous signals measured at the zinc absorption peak. J Microbiol. 51, 639–643 (2013). https://doi.org/10.1007/s12275-013-3412-2

Download citation

Keywords

  • Ferric uptake regulator
  • transcription regulator
  • crystallization
  • experimental phasing