Identification of the Zn2+ binding region in calreticulin.
Calreticulin binds Zn2+ with the relatively high affinity/low capacity. To determine the location of the Zn2+ binding site in calreticulin different domains of the protein were expressed in E. coli, using the glutathione S-transferase fusion protein system, and their Zn(2+)-dependent interaction with Zn(2+)-IDA-agarose were determined. Three distinct domains were used in this study: the N + P-domain (the first 290 residues); the N-domain (residues 1-182) and the proline-rich P-domain (residues 180-273). The N + P-domain bound to the Zn(2+)-IDA-agarose and were eluted with an increasing concentration of imidazole. The N-domain also bound 65Zn2+ as measured by the overlay method. The P-domain did not interact with the Zn(2+)-IDA-agarose and it did not bind any detectable amount of Zn2+. Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn2+ but they were modified by diethyl pyrocarbonate in the absence of Zn2+ suggesting that these residues may be involved in Zn2+ binding to calreticulin. We conclude that Zn2+ binding sites in calreticulin are localized to the N-domain of the protein, region that is not involved in Ca2+ binding to calreticulin.
MRC Group in Molecular Biology of Membrane, University of Alberta, Edmonton, Canada.
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Baksh
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Spamer
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Heilmann
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Michalak
D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D037282:Calreticulin; D002847:Chromatography, Agarose; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D037102:Lectins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D006639:Histidine; D015951:Factor Xa; D015032:Zinc; D004047:Diethyl Pyrocarbonate