TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain.
The lectin chaperone calreticulin (CRT) assists the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). It interacts with ERp57, a thiol-disulfide oxidoreductase that promotes the formation of disulfide bonds in glycoproteins bound by CRT. Here, we investigated the interaction between CRT and ERp57 by using biochemical techniques and NMR spectroscopy. We found that ERp57 binds to the P-domain of calreticulin, an independently folding domain comprising residues 189-288. Isothermal titration calorimetry showed that the dissociation constant of the CRT(189-288)/ERp57 complex is (9.1 +/- 3.0) x 10(-6) M at 8 degrees C. Transverse relaxation-optimized NMR spectroscopy provided data on the thermodynamics and kinetics of the complex formation and on the structure of this 66.5-kDa complex. The NMR measurements yielded a value of (18 +/- 5) x 10(-6) M at 20 degrees C for the dissociation constant and a lower limit for the first-order exchange rate constant of k(off) > 1,000 s(-1) at 20 degrees C. Chemical shift mapping showed that interactions with ERp57 occur exclusively through amino acid residues in the polypeptide segment 225-251 of CRT(189-288), which forms the tip of the hairpin structure of this domain. These results are analyzed with regard to the functional mechanism of the CRT/ERp57 chaperone system.
Frickel
Eva-Maria
EM
Institut für Biochemie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland.
Riek
Roland
R
Jelesarov
Ilian
I
Helenius
Ari
A
Wuthrich
Kurt
K
Ellgaard
Lars
L
D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D037282:Calreticulin; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human