Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system.
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.
Biochemistry Department, Faculty of Medicine, McGill University, Montréal, Québec, Canada.
Stephanie
S
Pollock
Guennadi
G
Kozlov
Marc-François
MF
Pelletier
Jean-François
JF
Trempe
Gregor
G
Jansen
Dimitri
D
Sitnikov
John J M
JJ
Bergeron
Kalle
K
Gehring
Irena
I
Ekiel
David Y
DY
Thomas
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D037281:Calnexin / Q000737:chemistry* / Q000378:metabolism*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D037281:Calnexin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases