Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system |
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Authors: | Pollock Stephanie Kozlov Guennadi Pelletier Marc-François Trempe Jean-François Jansen Gregor Sitnikov Dimitri Bergeron John J M Gehring Kalle Ekiel Irena Thomas David Y |
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Institution: | Biochemistry Department, Faculty of Medicine, McGill University, Montréal, Québec, Canada. |
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Abstract: | 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. |
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