MRC Group in Molecular Biology of Membrane, University of Alberta, Edmonton, Canada.
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.