Antonie van Leeuwenhoek

, 96:141

Biostructural analysis of the metal-sensor domain of CnrX from Cupriavidus metallidurans CH34

  • Guillaume Pompidor
  • Eric Girard
  • Antoine Maillard
  • Stéphanie Ramella-Pairin
  • Beate Bersch
  • Richard Kahn
  • Jacques Covès
Original Paper

Abstract

In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 Å using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P21, with unit cell parameters a = 31.87, b = 74.80, c = 93.67 Å, β = 90.107°. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.

Keywords

CnrX Cupriavidus metallidurans CH34 Extra-cytoplamic-function Heavy metal Signal transduction Structure 

References

  1. Bersch B, Favier A, Schanda P, van Aelst S, Vallaeys T, Covès J, Mergeay M, Wattiez R (2008) Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge. J Mol Biol 380:386–403PubMedCrossRefGoogle Scholar
  2. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50:760–763CrossRefGoogle Scholar
  3. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293PubMedCrossRefGoogle Scholar
  4. Ebel C (2007) Analytical ultracentrifugation. State of the art and perspectives. In: Uversky V, Permyakov EA (eds) Protein structures: methods in protein structure and stability analysis. Nova Science Publishers, New York, pp 229–260Google Scholar
  5. Franke S, Grass G, Rensing C, Nies DH (2003) Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. J Bacteriol 185:3804–3812PubMedCrossRefGoogle Scholar
  6. Geourjon C, Deléage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684PubMedGoogle Scholar
  7. Goldberg M, Pribyl T, Juhnke S, Nies DH (1999) Energetics and topology of CzcA, a cation/proton antiporter of the resistance-nodulation-cell division protein family. J Biol Chem 274:26065–26070PubMedCrossRefGoogle Scholar
  8. Grass G, Rensing C (2001) Genes involved in copper homeostasis in Escherichia coli. J Bacteriol 183:2145–2147PubMedCrossRefGoogle Scholar
  9. Grass G, Grosse C, Nies DH (2000) Regulation of the cnr cobalt and nickel resistance determinant from Ralstonia sp. strain CH34. J Bacteriol 182:1390–1398PubMedCrossRefGoogle Scholar
  10. Grass G, Fricke B, Nies DH (2005) Control of expression of a periplasmic nickel efflux pump by periplasmic nickel concentrations. Biometals 18:437–448PubMedCrossRefGoogle Scholar
  11. Grosse C, Friedrich S, Nies DH (2007) Contribution of extracytoplasmic function sigma factors to transition metal homeostasis in Cupriavidus metallidurans strain CH34. J Mol Microbiol Biotechnol 12:227–240PubMedCrossRefGoogle Scholar
  12. Jansson M, Li YC, Jendeberg L, Anderson S, Montelione BT, Nilsson B (1996) High level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7:131–141PubMedCrossRefGoogle Scholar
  13. Johnson BA (2004) Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol 278:313–352PubMedGoogle Scholar
  14. Kabsch W (1988) Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector. J Appl Cryst 21:916–924CrossRefGoogle Scholar
  15. Kim KH, Jung EJ, Im H, van der Lelie D, Kim EE (2008) Expression, purification, and crystallization and preliminary X-ray crystallographic analysis of CnrX from Cupriavidus metallidurans CH34. J Microbiol Biotechnol 18:43–47PubMedGoogle Scholar
  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  17. Liesegang H, Lemke K, Siddiqui RA, Schlegel HG (1993) Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34. J Bacteriol 175:767–778PubMedGoogle Scholar
  18. Lonetto MA, Brown KL, Rudd KE, Buttner MJ (1994) Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci USA 91:7573–7577PubMedCrossRefGoogle Scholar
  19. Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33:491–497PubMedCrossRefGoogle Scholar
  20. Mergeay M, Monchy S, Vallaeys T, Auquier V, Benotmane A, Bertin P, Taghavi S, Dunn J, van der Lelie D, Wattiez R (2003) Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS Microbiol Rev 27:385–410PubMedCrossRefGoogle Scholar
  21. Monchy S, Benotmane MA, Janssen P, Vallaeys T, Taghavi S, van der Lelie D, Mergeay M (2007) Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. J Bacteriol 189:7417–7425PubMedCrossRefGoogle Scholar
  22. Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339PubMedCrossRefGoogle Scholar
  23. Nies DH (2004) Incidence and function of sigma factors in Ralstonia metallidurans and other bacteria. Arch Microbiol 181:255–268PubMedCrossRefGoogle Scholar
  24. Pompidor G, Zoropogui A, Kahn R, Covès J (2007) Overproduction, purification and preliminary X-ray diffraction analysis of CzcE from Cupriavidus metallidurans CH34. Acta Crystallogr Sect F Struct Biol Cryst Commun 63:884–886PubMedCrossRefGoogle Scholar
  25. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modelling. Biophys J 78:1606–1619PubMedCrossRefGoogle Scholar
  26. Sendra V, Cannella D, Bersch B, Fieschi F, Ménage S, Lascoux D, Covès J (2006) CopH from Cupriavidus metallidurans CH34. A novel periplasmic copper-binding protein. Biochemistry 47:5557–5566CrossRefGoogle Scholar
  27. Tibazarwa C, Wuertz S, Mergeay M, Wyns L, van der Lelie D (2000) Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34. J Bacteriol 182:1399–1409PubMedCrossRefGoogle Scholar
  28. Zoropogui A, Gambarelli S, Covès J (2008) CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein. Biochem Biophys Res Commun 365:735–739PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Guillaume Pompidor
    • 1
  • Eric Girard
    • 1
  • Antoine Maillard
    • 1
  • Stéphanie Ramella-Pairin
    • 1
  • Beate Bersch
    • 2
  • Richard Kahn
    • 1
  • Jacques Covès
    • 1
  1. 1.Laboratoire des Protéines MembranairesInstitut de Biologie Structurale—Jean-Pierre Ebel, UMR 5075 CNRS-CEA-UJFGrenoble CedexFrance
  2. 2.Laboratoire de Résonance Magnétique NucléaireInstitut de Biologie Structurale—Jean-Pierre Ebel, UMR 5075 CNRS-CEA-UJFGrenoble CedexFrance

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