Identification of the Zn²⁺ binding region in calreticulin

Abstract

Calreticulin binds Zn 2+ with the relatively high affinit)/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 Zn2+-dependent interaction with Zn2+-lDA-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+-lDA-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 ZnZ+-IDA-agarose and it did not bind any detectable amount of Zu 2+. Chemical modification of calreticulin with diethyl pyrocarbonate indicated that rise 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 ealreticulin. 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.

Structural Basis of Carbohydrate Recognition by Calreticulin

“…CRT has also been reported to bind zinc, and four histidines within the lectin domain have been implicated in binding (40,43,(52)(53)(54)(55)(56) In conclusion, we have determined the structure of the CRT lectin domain in complex with its physiological ligand. The structure provides the framework for the design and interpretation of mutants that affect the multiple physiological functions of CRT.…”

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum

“…1) (Hammond et al, 1994;Ou et al, 1993;Spiro et al, 1996;Ware et al, 1995). They also bind to ATP, Ca 2+ , Zn 2+ and to one of the many thiol oxidoreductases of the ER, ERp57 (Baksh et al, 1995;Corbett et al, 2000;Oliver et al, 1997). Finally, they can associate with polypeptide segments of non-native glycoprotein conformers in a manner similar to other molecular chaperones, although the importance of this mode of association is controversial Saito et al, 1999).…”

“…In addition, Crt possesses multiple low-affinity (K d = 2 mM) Ca 2+ -binding sites that are involved in buffering ER Ca 2+ stores (Baksh and Michalak, 1991;Nakamura et al, 2001). Both chaperones bind Zn 2+ at sites within the globular domain (Baksh et al, 1995;Leach et al, 2002) and both bind ATP, although no ATPase activity has been detected. As is the case in other molecular chaperones, ATP may regulate conformational changes in Cnx and Crt.…”

Suppressive Roles of Calreticulin in Prostate Cancer Growth and Metastasis

“…6,16 -18 Other possible activities include integrin ␣-binding and cell adhesion, 19 -22 major histocompatibility class I assembly, 23 steroid-mediated gene regulation, 24 -26 as well as Zn 2ϩ binding and storage. 27 Gene knockout experiments further underscore the importance of this protein, as loss of calreticulin results in embryonic death from defective cardiac development. 28 Calreticulin consists of three distinctive domains.…”