Replacement of all alpha-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 alpha-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.
Cody
C W
CW
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205-2103.
Huang
P C
PC
D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D008239:Lysine / Q000235:genetics; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship
D005971:Glutamates; D002104:Cadmium; D018698:Glutamic Acid; D008668:Metallothionein; D008239:Lysine