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1: Proc Natl Acad Sci U S A 2002 Feb 12; [epub ahead of print]

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TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain.

Frickel EM, Riek R, Jelesarov I, Helenius A, Wuthrich K, Ellgaard L.

Institut fur Biochemie, Eidgenossische Technische Hochschule, CH-8092 Zurich, Switzerland; Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule, CH-8093 Zurich, Switzerland; and Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

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 [plus minus] 3.0) x 10([minus sign]6) M at 8iation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arC. 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 [plus minus] 5) x 10([minus sign]6) M at 20, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.blotting C for the dissociation constant and a lower limit for the first-order exchange rate constant of k(off) > 1,000 s([minus sign]1) at 20 in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO-1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO-1 by PAO and by heme may share some common pathwC. 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.

PMID: 11842220 [PubMed - as supplied by publisher]

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