Caenorhabditis elegans metallothionein isoform specificity - metal binding abilities and the role of histidine in CeMT1 and CeMT2
2009
Bofill, Roger | Orihuela, Rubén | Romagosa, Míriam | Domènech, Jordi | Atrian, Sílvia | Capdevila, Mercè
Two metallothionein (MT) isoforms have been identified in the model nematode Caenorhabditis elegans: CeMT1 and CeMT2, comprising two polypeptides that are 75 and 63 residues in length, respectively. Both isoforms encompass a conserved cysteine pattern (19 in CeMT1 and 18 in CeMT2) and, most significantly, as a result of their coordinative potential, CeMT1 includes four histidines, whereas CeMT2 has only one. In the present study, we present a comprehensive and comparative analysis of the metal [Zn(II), Cd(II) and Cu(I)] binding abilities of CeMT1 and CeMT2, performed through spectroscopic and spectrometric characterization of the recombinant metal-MT complexes synthesized for wild-type isoforms (CeMT1 and CeMT2), their separate N- and C-terminal moieties (NtCeMT1, CtCeMT1, NtCeMT2 and CtCeMT2) and a ΔHisCeMT2 mutant. The corresponding in vitro Zn/Cd- and Zn/Cu-replacement and acidification/renaturalization processes have also been studied, as well as protein modification strategies that make it possible to identify and quantify the contribution of the histidine residues to metal coordination. Overall, the data obtained in the present study are consistent with a scenario where both isoforms exhibit a clear preference for divalent metal ion binding, rather than for Cu coordination, although this preference is more pronounced towards cadmium for CeMT2, whereas it is markedly clearer towards Zn for CeMT1. The presence of histidines in these MTs is revealed to be decisive for their coordination performance. In CeMT1, they contribute to the binding of a seventh Zn(II) ion in relation to the M(II)₆-CeMT2 complexes, both when synthesized in the presence of supplemented Zn(II) or Cd(II). In CeMT2, the unique C-terminal histidine abolishes the Cu-thionein character that this isoform would otherwise exhibit.
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