Skip to main content
SpringerLink
Log in

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

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

Abstract

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M8 II–MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn6–MTC and Cd7–MTC species from M8 II–MTC after treatment with Chelex 100. The NMR characterization of Cd7–MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal–Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1–31) reveal a Cd3Cys9 cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32–64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu8–9–MTC). The subsequent generation of Cu12–MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.

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. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. González-Duarte P (2003) In: McCleverty J, Meyer TJ (eds) Comprehensive coordination chemistry II, vol 8. Elsevier-Pergamon, Amsterdam, pp 213–228

  2. Vašák M, Romero-Isart N (2006) In: King RB (ed) Encyclopedia of inorganic chemistry. Wiley, New York, pp 3208–3221

  3. Blindauer CA, Harrison MD, Parkinson JA, Robinson AK, Cavet JS, Robinson NJ, Sadler PJ (2001) Proc Natl Acad Sci USA 98:9593–9598

    Article  CAS  PubMed  Google Scholar 

  4. Peroza EA, Schmucki R, Guntert P, Freisinger E, Zerbe O (2009) J Mol Biol 387:207–218

    Article  CAS  PubMed  Google Scholar 

  5. Hong SH, Maret W (2003) Proc Natl Acad Sci USA 100:2255–2260

    Article  CAS  PubMed  Google Scholar 

  6. Gruber C, Sturzenbaum S, Gehrig P, Sack R, Hunziker P, Berger B, Dallinger R (2000) Eur J Biochem 267:573–582

    Article  CAS  PubMed  Google Scholar 

  7. Moltó E, Bonzón-Kulichenko E, Gallardo N, Andrés A (2007) Arch Biochem Biophys 467:31–40

    Article  PubMed  Google Scholar 

  8. Díaz S, Amaro F, Rico D, Campos V, Benítez L, Martín-González A, Hamilton EP, Orias E, Gutiérrez JC (2007) PLoS One. doi:10.1371/journal.pone.0000291

  9. Park H, Ahn IY, Choi HJ, Pyo SH, Lee HE (2007) Protein Exp Purif 52:82–88

    Article  CAS  Google Scholar 

  10. Jenny MJ, Warr GW, Ringwood AH, Baltzegar DA, Chapman RW (2006) Gene 379:156–165

    Article  CAS  PubMed  Google Scholar 

  11. Syring RA, Brouwer-Hoexum T, Brouwer M (2000) Comp Biochem Physiol C Toxicol Pharmacol 125:325–332

    CAS  PubMed  Google Scholar 

  12. García S, Prado M, Degano R, Domínguez A (2002) J Biol Chem 277:37359–37368

    Article  PubMed  Google Scholar 

  13. Domènech J, Bofill R, Tinti A, Torreggiani A, Atrian S, Capdevila M (2008) Biochim Biophys Acta 1784:693–704

    PubMed  Google Scholar 

  14. Narula SS, Brouwer M, Hua Y, Armitage IM (1995) Biochemistry 34:620–631

    Article  CAS  PubMed  Google Scholar 

  15. Muñoz A, Forsterling FH, Shaw CF III, Petering DH (2002) J Biol Inorg Chem 7:713–724

    Article  PubMed  Google Scholar 

  16. Brouwer M, Brouwer-Hoexum T (1998) Arch Biochem Biophys 351:257–264

    Article  CAS  PubMed  Google Scholar 

  17. Yudkovski Y, Shechter A, Chalifa-Caspi V, Auslander M, Ophir R, Dauphin-Villemant C, Waterman M, Sagi A, Tom M (2007) Insect Mol Biol 16:661–674

    CAS  PubMed  Google Scholar 

  18. Cols N, Romero-Isart N, Capdevila M, Oliva B, González-Duarte P, González-Duarte R, Atrian S (1997) J Inorg Biochem 68:157–166

    Article  CAS  PubMed  Google Scholar 

  19. Valls M, Bofill R, González-Duarte R, González-Duarte P, Capdevila M, Atrian S (2001) J Biol Chem 276:32835–32843

    Article  CAS  PubMed  Google Scholar 

  20. Capdevila M, Cols N, Romero-Isart N, González-Duarte R, Atrian S, González-Duarte P (1997) Cell Mol Life Sci 53:681–688

    Article  CAS  PubMed  Google Scholar 

  21. Capdevila M, Domenech J, Pagani A, Tio L, Villarreal L, Atrian S (2005) Angew Chem Int Ed 44:4618–4622

    Article  CAS  Google Scholar 

  22. Birchmeier W, Christen P (1971) FEBS Lett 18:209–213

    Article  CAS  PubMed  Google Scholar 

  23. Overnell J, Good M, Vašák M (1988) Eur J Biochem 172:171–177

    Article  CAS  PubMed  Google Scholar 

  24. Brouwer M, Syring R, Brouwer-Hoexum T (2002) J Inorg Biochem 88:228–239

    Article  CAS  PubMed  Google Scholar 

  25. Brouwer M, Schlenk D, Ringwood AH, Brouwer-Hoexum T (1992) Arch Biochem Biophys 294:461–468

    Article  CAS  PubMed  Google Scholar 

  26. Pagani A, Villarreal L, Capdevila M, Atrian S (2007) Mol Microbiol 63:256–269

    Article  CAS  PubMed  Google Scholar 

  27. Tio L, Villarreal L, Atrian S, Capdevila M (2004) J Biol Chem 279:24403–24413

    Article  CAS  PubMed  Google Scholar 

  28. Brouwer M (1996) Adv Inorg Biochem 11:235–260

    CAS  Google Scholar 

  29. Dance IG, Fisher K, Lee G (1992) In: Stillman MJ, Shaw III CF, Suzuki KT (eds) Metallothioneins. VCH, New York, pp 284–345

  30. Calderone V, Dolderer B, Hartmann HJ, Echner H, Luchinat C, Del Bianco C, Mangani S, Weser U (2005) Proc Natl Acad Sci USA 102:51–56

    Article  CAS  PubMed  Google Scholar 

  31. Dallinger R, Chabicovsky M, Hodl E, Prem C, Hunziker P, Manzl C (2005) Am J Physiol Regul Integr Comp Physiol 289:R1185–R1195

    CAS  PubMed  Google Scholar 

  32. Jensen LT, Peltier JM, Winge D (1998) J Biol Inorg Chem 3:627–631

    Article  CAS  Google Scholar 

  33. Zhang L, Pickering IJ, Winge DR, George GN (2008) Chem Biodivers 5:2042–2049

    Article  CAS  PubMed  Google Scholar 

  34. Roschitzki B, Vašák M (2002) J Biol Inorg Chem 7:611–616

    Article  CAS  PubMed  Google Scholar 

  35. Pountney DL, Schauwecker I, Zarn J, Vašák M (1994) Biochemistry 33:9699–9705

    Article  CAS  PubMed  Google Scholar 

  36. Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008) Nat Methods 5:135–146

    Article  PubMed  Google Scholar 

  37. Henehan CJ, Pountney DL, Zerbe O, Vašák M (1993) Protein Sci 2:1756–1764

    Article  CAS  PubMed  Google Scholar 

  38. Meloni G, Zovo K, Kazantseva J, Palumaa P, Vašák M (2006) J Biol Chem 281:14588–14595

    Article  CAS  PubMed  Google Scholar 

  39. Peroza EA, Kaabi AA, Meyer-Klaucke W, Wellenreuther G, Freisinger E (2009) J Inorg Biochem 103:342–353

    Article  CAS  PubMed  Google Scholar 

  40. Vašák M, Kagi JHR (1983) In: Sigel H (ed) Spectroscopic properties of metallothioneins. Metal ions in biological systems, vol 15. Dekker, New York, pp 213–273

  41. Hasler DW, Jensen LT, Zerbe O, Winge DR, Vašák M (2000) Biochemistry 39:14567–14575

    Article  CAS  PubMed  Google Scholar 

  42. Wang Y, Mackay EA, Kurasaki M, Kagi JH (1994) Eur J Biochem 225:449–457

    Article  CAS  PubMed  Google Scholar 

  43. Willner H, Vašák M, Kagi JH (1987) Biochemistry 26:6287–6292

    Article  CAS  PubMed  Google Scholar 

  44. Vašák M (1991) In: Riordan JF, Vallee BL (eds) Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods in enzymology, vol 205, metallobiochemistry. Academic Press, San Diego, pp 452–458

  45. Bongers J, Walton CD, Richardson DE, Bell JU (1988) Anal Chem 60:2683–2686

    Article  CAS  PubMed  Google Scholar 

  46. Fabris D, Zaia J, Hathout Y, Fenselau C (1996) J Am Chem Soc 118:12242–12243

    Article  CAS  Google Scholar 

  47. Gans P, Sabatini A, Vacca A (1999) Ann Chim 89:45–49

    CAS  Google Scholar 

  48. Bofill R, Palacios O, Capdevila M, Cols N, González-Duarte R, Atrian S, González-Duarte P (1999) J Inorg Biochem 73:57–64

    Article  CAS  PubMed  Google Scholar 

  49. Donaire A, Zhou ZH, Adams MM, La Mar GN (1996) J Biomol NMR 7:35–47

    Article  CAS  PubMed  Google Scholar 

  50. Goddard TD, Kneller DG SPARKY 3—NMR assignment program. University of California, San Francisco

Download references

Acknowledgments

The authors gratefully acknowledge financial support from DURSI, Generalitat de Catalunya (SGR2009-68), Fundación Séneca de la Región de Murcia (05716/PI/07), and the Spanish Ministerio de Ciencia e Innovación (CTQ2008-02767/BQU). L.A.A is indebted to the Program Juan de la Cierva (Ministerio de Ciencia e Innovación, Spain). We are grateful to Marius Brouwer (Institute of Marine Sciences, University of Southern Mississippi, USA) who kindly provided the C. sapidus MTC cDNA clone. The authors thank the NMR Facility of the Serveis Cientificotècnics of the Universitat de Barcelona for providing access to the 800-MHz NMR spectrometer.

Author information

Authors and Affiliations

  1. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain

    Montserrat Serra-Batiste & Pilar González-Duarte

  2. Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028, Barcelona, Spain

    Neus Cols

  3. Departamento de Química Física, Facultad de Ciencias, Universidad de Alicante, 03080, Alicante, Spain

    Luis A. Alcaraz

  4. Departamento de Química Inorgánica, Facultad de Química, Universidad de Murcia, 30100, Murcia, Spain

    Antonio Donaire

  5. Department of Biochemistry, University of Zurich, 8057, Zurich, Switzerland

    Milan Vašák

Authors
  1. Montserrat Serra-Batiste
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Neus Cols
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luis A. Alcaraz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonio Donaire
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pilar González-Duarte
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Milan Vašák
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Pilar González-Duarte or Milan Vašák.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 560 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Serra-Batiste, M., Cols, N., Alcaraz, L.A. et al. The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets. J Biol Inorg Chem 15, 759–776 (2010). https://doi.org/10.1007/s00775-010-0644-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-010-0644-z

Keywords

Search

Navigation