Replacement of All α-domain lysines with glutamates reduces metallothionein detoxification function

Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, including three in each of two metal binding domains. We used site-directed mutagenesis to replace these intradomain lysines in Chinese hamster ovary MT2 with glutamic acid and/or glutamine. These mutant MTs were expressed in a metal sensitive yeast host. One mutant which had all three lysines in the α-domain replaced by glutamates (K43,51,56E) exhibited a reduced ability, relative to native MT, to protect yeast transformants against otherwise toxic levels of cadmium. This triply substituted mutant also exhibited anomalous migration on a non-denaturing gel relative to wild type MT and other MT lysine mutants, suggesting that the intradomain lysines are important in maintaining the conformational integrity of MT.