Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character

After the resolution of the 3D structure of the Cd₉-aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M₉-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M₆-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu₄-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.