JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 5, pp 759–776

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

DOI: 10.1007/s00775-010-0644-z

Cite this article as:
Serra-Batiste, M., Cols, N., Alcaraz, L.A. et al. J Biol Inorg Chem (2010) 15: 759. doi:10.1007/s00775-010-0644-z

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 M8II–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 M8II–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

MetallothioneinMetal–thiolate clustersZincCadmiumCopper

Supplementary material

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

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