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 function 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. When both lysines were replaced by glutamine (K30,31Q), a reduction in the ability of MT to protect yeast transformants 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 charged amino acid must be present in the hinge for the proper expression of MT.
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2103.
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Huang
D000818:Animals; D016466:CHO Cells; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D005973:Glutamine; D008658:Inactivation, Metabolic; D008239:Lysine; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000493:pharmacokinetics; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D008670:Metals; D012333:RNA, Messenger; D002104:Cadmium; D005973:Glutamine; D008668:Metallothionein; D008239:Lysine