Substitution of glutamic acids for the conserved lysines in the alpha domain affects metal binding in both the alpha and beta domains of 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 assayed with, expressed in and purified from a cadmium sensitive strain of yeast Saccharomyces cerevisiae. Circular dichroism analyses of the mutated protein, mutein K43,51,56E, revealed that the overall structure remained unchanged. However, a 1-D 113Cd NMR study detected significant differences in the chemical shifts of the corresponding resonances between wild type protein and the recombinant mutein. Reduction of integrated 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 metals in the beta-domain of the mutein was also observed, which was unexpected 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 recombinant 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 stability of both metal-binding clusters, a feature critical to metal detoxification.
College of Life Science, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.
P K
PK
Pan
F Y
FY
Hou
C W
CW
Cody
P C
PC
Huang
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000633:toxicity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism
D005971:Glutamates; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D008239:Lysine