Identification of the Zn2+ binding region in calreticulin

FEBS Letters

Spamer, Cornelia¹  Heilmann, Claus¹  Baksh, Shairaz²  Michalak, Marek² 

[1] Department of Gastroenterology, University of Freiburg, D-7800 Freiburg, Germany;MRC Group in Molecular Biology of Membrane and the Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada

关键词: Calreticulin;    Zinc binding;    Endoplasmic reticulum;    GST;    glutathione S-transferase;    IDA;    iminodiacetate-substituted agarose;    DEPC;    diethyl pyrocarbonate;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    MOPS;    4-morpholinepropanesulfonic acid;   

DOI  :  10.1016/0014-5793(95)01246-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown