Bioactivity of Metallothionein-3 Correlates with its Novel β Domain Sequence Rather than Metal Binding Properties

Human and mouse metallothionein-3 (MT-3) molecules exhibit the same metal binding stoichiometry with Zn(II), Cd(II), or Cu(I) as MT-1 or MT-2 molecules, suggesting that MT-3 consists of two domains enfolding separate polymetallic clusters. The kinetic reactivities of Zn(II) complexes of MT-3 with the chelator ethylenediaminetetraacetic acid (EDTA) or the thiol reagent dithiobis(2-nitrobenzoic acid) (DTNB) resembles the reactivity of ZnMT-1. Furthermore, the candidate α and β domain peptides of human MT-3 are very similar to MT-1 domain peptides in the reactivity of Zn(II) complexes. Zn(II) complexes of human and mouse MT-3 inhibit the survival of rat cortical neurons cultured in the presence of an Alzheimer's disease brain extract. Inhibitory activity is unique to the MT-3 isoform and is a property of the N-terminal β domain. The inhibitory activity of the 32-residue MT-3 β domain is abolished by a double mutation within the β domain resulting in the conversion of the C-P-C-P sequence to either C-SC-A or C-T-C-T. Thus, the bioactivity arises from a novel structure of the N-terminal β domain of MT-3 and not any unusual metal-binding properties.