Metallothionein detoxification function is impaired by replacement of both conserved lysines with glutamines in the hinge between the two domains

Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, two of which constitute the hinge between two metal binding domains. By site-directed mutagenesis and recombinant DNA techniques, we replaced the interdomain lysines in Chinese hamster ovary MT2 with all possible combinations of glutamic acid and/or glutamine. The resultant MTs were expressed and assayed for detoxification feunction in a transformed yeast system. Results showed that these mutant MTs, like the native protein, bound seven atoms of divalent metal per molecule and conferred cadmium resistance to a metal-sensitive yeast host. Replacement of one or both of the lysines in the interdomain region was inconsequential to the structure and function of MT, unless both substituted residues were uncharged