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

  • Montserrat Serra-Batiste
  • Neus Cols
  • Luis A. Alcaraz
  • Antonio Donaire
  • Pilar González-Duarte
  • Milan Vašák
Original Paper

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.

Keywords

Metallothionein Metal–thiolate clusters Zinc Cadmium Copper 

Notes

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.

Supplementary material

775_2010_644_MOESM1_ESM.pdf (560 kb)
Supplementary material 1 (PDF 560 kb)

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Copyright information

© SBIC 2010

Authors and Affiliations

  • Montserrat Serra-Batiste
    • 1
  • Neus Cols
    • 2
  • Luis A. Alcaraz
    • 3
  • Antonio Donaire
    • 4
  • Pilar González-Duarte
    • 1
  • Milan Vašák
    • 5
  1. 1.Departament de Química, Facultat de CiènciesUniversitat Autònoma de BarcelonaBellaterra, BarcelonaSpain
  2. 2.Departament de Genètica, Facultat de BiologiaUniversitat de BarcelonaBarcelonaSpain
  3. 3.Departamento de Química Física, Facultad de CienciasUniversidad de AlicanteAlicanteSpain
  4. 4.Departamento de Química Inorgánica, Facultad de QuímicaUniversidad de MurciaMurciaSpain
  5. 5.Department of BiochemistryUniversity of ZurichZurichSwitzerland

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