SUBSTITUTION OF GLUTAMIC ACIDS FOR THE CONSERVED LYSINES IN THE ALPHA-DOMAIN AFFECTS METAL-BINDING IN BOTH THE ALPHA-DOMAIN AND BETA-DOMAINOF MAMMALIAN METALLOTHIONEIN

Lysine residues are highly conserved in mammalian metallothioneins(MTs ). Recombinant mutant Chinese hamster MT2 in which all of the lysines (K) in the alpha-domain were substituted by glutamic acids (E) was ass ayed with, expressed in and purified from a cadmium sensitive strain o f yeast Saccharomyces cerevisiae. Circular dichroism analyses of the m utated protein, mutein K43,51,56E, revealed that the overall structure remained unchanged. However, a 1-D Cd-113 NMR study detected signific ant differences in the chemical shifts of the corresponding resonances between wild type protein and the recombinant mutein. Reduction of in tegrated intensity in the NMR spectra was also observed for resonances from the four-metal cluster (I and V-VII) in the alpha-domain of the mutein. At various temperatures, facile intermolecular exchange of met als in the beta-domain of the mutein was also observed, which was unex pected and was different from wild type. Our results thus demonstrate that replacing all three lysines by glutamic acids in the alpha-domain changed metal-thiolate interactions in both domains of the recombinan t mutein. This may explain the reduced ability of the mutein to convey cadmium resistance. We propose that while the lysine residues in the alpha-domain of wild type MT are not critical for maintaining protein structure, they play a role in regulating the microenvironment and sta bility of both metal-binding clusters, a feature critical to metal det oxification. (C) 1994 Academic Press, Inc.