Skip to main content

Advertisement

SpringerLink
Log in

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34

  • Original Article
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Δa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTCXXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl–methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. 15N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first β-strand and the first α-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2A–F
Fig. 3
Fig. 4
Fig. 5
Fig. 6A, B
Fig. 7

Similar content being viewed by others

Abbreviations

CCA:

α-cyano-4-hydroxy-trans-cinnamic acid

CSI:

chemical shift index

HSQC:

1H-detected heteronuclear single-quantum coherence

MerAa:

the 68 amino acid N-terminal extension of the mercuric reductase

NOE:

nuclear Overhauser effect

RDCs:

residual dipolar couplings

TCEP-HCl:

tris(2-carboxyethyl)phosphine hydrochloride

References

  1. Silver S, Phung LT (1996) Annu Rev Microbiol 50:753–789

    CAS  PubMed  Google Scholar 

  2. Hobman JL, Brown NL (1997) Metal Ions Biol Syst 34:527–568

    CAS  Google Scholar 

  3. Liebert CA, Hall RM, Summers AO (1999) Microbiol Mol Biol Rev 63:507–522

    CAS  PubMed  Google Scholar 

  4. Barkay T, Miller SM, Summers AO (2003) FEMS Microbiol Rev 27:355–384

    Article  CAS  PubMed  Google Scholar 

  5. O’Halloran TV, Walsh CT (1987) Science 235:211–214

    CAS  PubMed  Google Scholar 

  6. Brown NL, Stoyanov JV, Kidd SP, Hobman JL (2003) FEMS Microbiol Rev 27:145–163

    Article  CAS  PubMed  Google Scholar 

  7. Sahlman L, Jonsson B-H (1992) Eur J Biochem 205:375–381

    CAS  PubMed  Google Scholar 

  8. Brown NL, Camakaris J, Lee BT, Williams T, Morby AP, Parkhill J, Rouch DA (1991) J Cell Biochem 46:106–114

    CAS  PubMed  Google Scholar 

  9. Fox B, Walsh CT (1982) J Biol Chem 257:2498–2503

    CAS  PubMed  Google Scholar 

  10. Moore MJ, Walsh CT (1989) Biochemistry 28:1183–1194

    CAS  PubMed  Google Scholar 

  11. Brown NL, Shih Y-C, Leang C, Glendinning KJ, Hobman JL, Wilson JR (2002) Biochem Soc Trans 30:715–718

    CAS  PubMed  Google Scholar 

  12. Sedlmeier R, Altenbuchner J (1992) Mol Gen Genet 236:76–85

    CAS  PubMed  Google Scholar 

  13. Shiering N, Kabsch W, Moore MJ, Distefano MD, Walsh CT, Pai EF (1991) Nature 352:168–172

    CAS  PubMed  Google Scholar 

  14. Steele RA, Opella SJ (1997) Biochemistry 36:6885–6895

    Article  CAS  PubMed  Google Scholar 

  15. Qian H, Sahlman L, Eriksson P-O, Hambraeus C, Edlund U, Sethson I (1998) Biochemistry 37:9316–9322

    Article  CAS  PubMed  Google Scholar 

  16. Opella SJ, Desilva TM, Veglia G (2002) Curr Opin Chem Biol 6:217–223

    Article  CAS  PubMed  Google Scholar 

  17. Arnesano F, Banci L, Bertini I, Ciofi-Baffoni S, Molteni E, Huffman DL, O’Halloran TV (2002) Genome Res 12:255–271

    Article  CAS  PubMed  Google Scholar 

  18. Mergeay M, Monchy S, Vallaeys T, Auquier V, Benotmane A, Bertin P, Taghavi S, Dunn J, van der Lelie D, Wattiez R (2003) FEMS Microbiol Rev 27:385–410

    Article  CAS  PubMed  Google Scholar 

  19. Banci L, Bertini I, Ciofi-Baffoni S, Huffman DL, O’Halloran TV (2001) J Biol Chem 276:8415–8426

    Article  CAS  PubMed  Google Scholar 

  20. Bersch B, Rossy E, Covès J, Brutscher B (2003) J Biomol NMR 27:57–67

    Article  CAS  PubMed  Google Scholar 

  21. Laemmli UK (1970) Nature 227:680–685

    PubMed  Google Scholar 

  22. Zhu G, Bax A (1990) J Magn Reson 101:114–119

    Google Scholar 

  23. Grzesiek S, Anglister J, Bax A (1993) J Magn Reson B101:114–119

    Google Scholar 

  24. Bax A, Mehlkopf AF, Smidt J (1979) J Magn Reson 35:167–169

    CAS  Google Scholar 

  25. Rückert M, Otting G (2000) J Am Chem Soc 122:7793–7797

    Article  Google Scholar 

  26. Farrow NA, Muhandiram R, Singer AU, Pascal SM, Kay CM, Gish, G, Shoelson SE, Pawson T, Forman-Kay JD, Kay LE (1994) Biochemistry 34:868–878

    Google Scholar 

  27. Tsan P, Hus J-C, Caffrey M, Marion D, Blackledge M (2000) J Am Chem Soc 121:2311–2312

    Google Scholar 

  28. Blackledge M, Medvedeva S, Poncin M, Guerlesquin F, Bruschi M, Marion D (1995) J Mol Biol 245:661–681

    Article  CAS  PubMed  Google Scholar 

  29. Hus J-C, Marion D, Blackledge M (2000) J Mol Biol 298:927–936

    Google Scholar 

  30. Sibille N, Pardi A, Simorre J-P, Blackledge M (2001) J Am Chem Soc 49:12135–12146

    Article  Google Scholar 

  31. Gurd F (1972) Methods Enzymol 25:424–438

    CAS  Google Scholar 

  32. Metzler WJ, Wittekind M, Goldfarb V, Mueller L, Farmer BT (1996) J Am Chem Soc 119:6800–6801

    Article  Google Scholar 

  33. Smith B, Ito Y, Raine A, Teichmann S, Ben-Tovim L, Nietlispach D, Broadhurst RW, Terada T, Kelly M, Oschkinat H, Shibata T, Yokoyama S, Laue E (1996) J Biomol NMR 8:360–369

    CAS  Google Scholar 

  34. Gardner K, Rosen MK, Kay LE (1997) Biochemistry 36:1389–1401

    Article  CAS  PubMed  Google Scholar 

  35. Gardner K, Kay LE (1998) Annu Rev Biophys Biomol Struct 27:357–406

    Article  CAS  PubMed  Google Scholar 

  36. Banci L, Bertini I, Ciofi-Baffoni S, Finney LA, Outten CE, O’Halloran TV (2002) J Mol Biol 323:883–897

    Article  CAS  PubMed  Google Scholar 

  37. Lipari G, Szabo A (1982a) J Am Chem Soc 104:4546–4559

    Google Scholar 

  38. Lipari G, Szabo A (1982a) J Am Chem Soc 104:4559–4570

    Google Scholar 

  39. Dosset P, Hus J-C, Blackledge M, Marion D (2000) J Biomol NMR 16:23–28

    Article  CAS  PubMed  Google Scholar 

  40. Lu W, Zelazowski AJ, Stillman MJ (1993) Inorg Chem 32:919–926

    CAS  Google Scholar 

  41. Lu W, Stillman MJ (1993) J Am Chem Soc 115:3291–3299

    CAS  Google Scholar 

  42. Kihlken MA, Leech AP, Le Brun NE (2002) Biochem J 368:729–739

    Article  CAS  PubMed  Google Scholar 

  43. Morby AP, Hobman JL, Brown NL (1995) Mol Microbiol 17:25–35

    CAS  PubMed  Google Scholar 

  44. DeSilva T, Veglia G, Porcelli F, Pranter AM, Opella SJ (2002) Biopolymers 64:189–197

    Article  CAS  PubMed  Google Scholar 

  45. Guex N, Peitsch MC (1997) Electrophoresis 1:2714–2723

    Google Scholar 

Download references

Acknowledgements

We are very grateful to David Lemaire and David Lascoux for their help in mass spectrometry experiments, to Jean-Pierre Andrieu for N-terminal sequencing, to Elisabeth Mintz and Vincent Forge for the access to the CD apparatus, the use of the SPECFIT software, and for fruitful discussions. Finally, Tatiana Valleys and Max Mergeay are acknowledged for constant encouragements. E.R. and L.C. were supported by PhD grants from the Région Rhône-Alpes and the Commissariat à l’Energie Atomique, respectively.

Author information

Author notes

  1. Jacques Covès

    Present address: IBS/LPM, 41 Rue Jules Horowitz, 38027, Grenoble Cedex, France

Authors and Affiliations

  1. Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, CEA-Grenoble, DRDC/CB, UMR 5047 CNRS – CEA, Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Cedex 9, Grenoble , France

    Emmanuel Rossy, Ludovic Champier & Jacques Covès

  2. Laboratoire de Résonance Magnétique Nucléaire, Institut de Biologie Structurale – Jean-Pierre Ebel, UMR 5075 CNRS – CEA, Université Joseph Fourier, 41 Rue Jules Horowitz, 38027, Grenoble Cedex, France

    Beate Bersch, Bernhard Brutscher & Martin Blackledge

Authors
  1. Emmanuel Rossy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ludovic Champier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beate Bersch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bernhard Brutscher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin Blackledge
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jacques Covès
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Beate Bersch or Jacques Covès.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rossy, E., Champier, L., Bersch, B. et al. Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34. J Biol Inorg Chem 9, 49–58 (2004). https://doi.org/10.1007/s00775-003-0495-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-003-0495-y

Keywords

Search

Navigation