title: NMR structures of 36 and 73-residue fragments of the calreticulin P-domain.
abstract: Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189-288. This structure shows an extended hairpin topology, with three short anti-parallel beta-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225-251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221-256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189-288). Of the 36 residues present in CRT(221-256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221-256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221-256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189-261), in which the tip of the hairpin and the first beta-sheet are well structured, but the residues 189-213 are disordered, presumably due to lack of stabilizing interactions across the hairpin.
authors.affiliation: Institut für Biochemie, Eidgenössische Technische Hochschule Zürich, Switzerland.
authors.forename: Lars
authors.initials: L
authors.lastname: Ellgaard
authors.forename: Pascal
authors.initials: P
authors.lastname: Bettendorff
authors.forename: Daniel
authors.initials: D
authors.lastname: Braun
authors.forename: Torsten
authors.initials: T
authors.lastname: Herrmann
authors.forename: Francesco
authors.initials: F
authors.lastname: Fiorito
authors.forename: Ilian
authors.initials: I
authors.lastname: Jelesarov
authors.forename: Peter
authors.initials: P
authors.lastname: Güntert
authors.forename: Ari
authors.initials: A
authors.lastname: Helenius
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037282:Calreticulin / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D015870:Gene Expression; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D012996:Solutions
chemical_list: D037282:Calreticulin; D018832:Molecular Chaperones; D010446:Peptide Fragments; D012996:Solutions