METALLOTHIONEIN DETOXIFICATION FUNCTION IS IMPAIRED BY REPLACEMENT OFBOTH CONSERVED LYSINES WITH GLUTAMINES IN THE HINGE BETWEEN THE 2 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 resu ltant 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 a nd conferred cadmium resistance to a metal-sensitive yeast host. Repla cement of one or both of the lysines in the interdomain region was inc onsequential to the structure and function of MT, unless both substitu ted residues were uncharged. When both lysines were replaced by glutam ine (K30,31Q), a reduction in the ability of MT to protect yeast trans formants against otherwise toxic levels of cadmium was observed. This diminished metal detoxification capacity was due to a decrease in the steady-state level of MT. These results suggest that at least one char ged amino acid must be present in the hinge for the proper expression of MT.