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Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two‐hybrid system
Author(s) -
Pollock Stephanie,
Kozlov Guennadi,
Pelletier MarcFrançois,
Trempe JeanFrançois,
Jansen Gregor,
Sitnikov Dimitri,
Bergeron John JM,
Gehring Kalle,
Ekiel Irena,
Thomas David Y
Publication year - 2004
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7600119
Subject(s) - calnexin , endoplasmic reticulum , protein disulfide isomerase , chemistry , folding (dsp implementation) , nuclear magnetic resonance spectroscopy , domain (mathematical analysis) , biophysics , biochemistry , microbiology and biotechnology , biology , stereochemistry , calreticulin , engineering , mathematical analysis , mathematics , electrical engineering
Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P‐domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin‐like repeats and a short basic C‐terminal tail. Here we show direct interactions between the tip of the calnexin P‐domain and the ERp57 basic C‐terminus by using NMR and a novel membrane yeast two‐hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P‐domain is active in binding ERp57, and we determine the structure of the bound conformation of the P‐domain peptide. The experimental strategy of using the MYTHS two‐hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.