Premium
Identification of the Zn 2+ binding region in calreticulin
Author(s) -
Baksh Shairaz,
Spamer Cornelia,
Heilmann Claus,
Michalak Marek
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01246-4
Subject(s) - calreticulin , chemistry , binding site , agarose , binding domain , imidazole , binding protein , biochemistry , endoplasmic reticulum , gene
Calreticulin binds Zn 2+ with the relatively high affinity/low capacity. To determine the location of the Zn 2+ 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 65 Zn 2+ 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 Zn 2+ . Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn 2+ but they were modified by diethyl pyrocarbonate in the absence of Zn 2+ suggesting that these residues may be involved in Zn 2+ binding to calreticulin. We conclude that Zn 2+ binding sites in calreticulin are localized to the N‐domain of the protein, region that is not involved in Ca 2+ binding to calreticulin.