The requirements for stable metallothionein clusters examined using synthetic lobster domains

Metallothioneins (MTs) are small, cysteine-rich proteins which detoxify xenobiotic metals such as cadmium (Cd) and mercury (Hg). In crustaceans and mammals they consist of two independent domains which are folded around metal-thiolate clusters. MT clusters of different origins, exhibiting distinct, highly conserved cysteine positions on their sequences, show differences in metal-cysteine coordination and reactivity. Lobster-MT, containing two Cd3β domains, is an important model for structure–function relationships among the clusters. The influence of (1) the position of the cysteine residues and (2) steric and electrostatic effects of neighboring amino acids on the folding and stability of MT cluster were investigated. Thus, the native lobster βC and βN domains (each having nine cysteines and binding three M2+ ions) and a modified domain Cd3βC→N, in which the cysteines of the C-terminal domain were relocated to match the positions of those in the N-terminal domain, were chemically prepared and characterized. The synthetic native domains (Cd3βC and Cd3βN) were found to exhibit spectroscopic properties, metal-binding affinities and kinetic reactivity similar to the holo-protein. However, the modified Cd3βC→N domain was unusually reactive and in the presence of Chelex, metal chelation resin, aggregated to a Cd5(βC→N)2 dimer, which exhibited unusual structure as observed by its 113Cd-nuclear magnetic resonance. These differences in structure and reactivity demonstrated that the requirements for formation of a stable Cd3S9 β-cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and must include interactions involving neighboring, non-cysteine amino acids.