Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system

EMBO J. 2004 Mar 10;23(5):1020-9. doi: 10.1038/sj.emboj.7600119. Epub 2004 Feb 26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calnexin / chemistry*
  • Calnexin / metabolism*
  • Dogs
  • Endoplasmic Reticulum / metabolism*
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism*
  • Intracellular Membranes / chemistry
  • Intracellular Membranes / metabolism
  • Isomerases / chemistry*
  • Isomerases / genetics
  • Isomerases / metabolism*
  • 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
  • Two-Hybrid System Techniques*


  • Heat-Shock Proteins
  • Peptide Fragments
  • Calnexin
  • Isomerases
  • Protein Disulfide-Isomerases