1. Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system.
- Author
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Pollock S, Kozlov G, Pelletier MF, Trempe JF, Jansen G, Sitnikov D, Bergeron JJ, Gehring K, Ekiel I, and Thomas DY
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Dogs, Heat-Shock Proteins genetics, Intracellular Membranes chemistry, Intracellular Membranes metabolism, Isomerases genetics, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Binding, Protein Disulfide-Isomerases metabolism, Protein Structure, Tertiary, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Calnexin chemistry, Calnexin metabolism, Endoplasmic Reticulum metabolism, Heat-Shock Proteins chemistry, Heat-Shock Proteins metabolism, Isomerases chemistry, Isomerases metabolism, Two-Hybrid System Techniques
- 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.
- Published
- 2004
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