Identification of the Zn2+ binding region in calreticulin

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Abstract
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 Zn2+‐dependent interaction with Zn2+‐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 Zn2+‐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 Zn2+‐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.