Metal exchange in metallothioneins -: a novel structurally significant Cd5 species in the alpha domain of human metallothionein 1a

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn2+ ions for Cd2+ ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd2+ ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd2+ ion to form a novel Cd-5 alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd-5 alpha species following addition of 5.0 molar equivalents of Cd2+ to a solution of Cd-4 alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd-5 alpha species in contrast to the derivative-shaped spectrum of the Cd-4 alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd2+ ions in the Cd-4 alpha species. The Cd-113 chemical shift of the fifth Cd2+ is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd2+ ions in Cd-4 alpha-MT by both direct and indirect Cd-113 NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect H-1-Cd-113 HSQC NMR spectra suggests that the coordination environment of the additional Cd2+ is not tetrahedral to four thiolates, as is the case with the four Cd2+ ions in the Cd-4 alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.