PDBsum entry 1k9c

Metal transport

PDB id

1k9c

Contents

			Protein chain

					74 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Nmr structures of 36 and 73-Residue fragments of the calreticulin p-Domain.

Authors

L.Ellgaard, P.Bettendorff, D.Braun, T.Herrmann, F.Fiorito, I.Jelesarov, P.Güntert, A.Helenius, K.Wüthrich.

Ref.

J Mol Biol, 2002, 322, 773-784. [DOI no: 10.1016/S0022-2836(02)00812-4]

PubMed id

12270713

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.



	Figure 4. Figure 4. Diagonal plots of the NOE upper distance constraints identified in CRT(221-256) (a) and CRT(189-261) (b). The sequence numbering is shown on both axes. The presence of a distance constraint between a pair of residues is indicated by a square. Increasing darkness of the squares indicates an increasing number of NOE constraints between the two residues, with black squares representing five or more NOEs. No distinction is made between NOEs involving backbone or side-chain hydrogen atoms. In (b), the region corresponding to CRT(221-256) is indicated by broken lines.		Figure 5. Figure 5. (a) and (b) Bundles of the 20 energy-minimized conformers used to represent the NMR structure of CRT(221-256) after superposition for best fit of the backbone atoms N, C^a and C' of the residues 223-254. (a) All-heavy-atom presentation of the complete structure. The backbone is colored green, positively charged residues are blue, negatively charged residues are red, and hydrophobic and polar residues are white. (b) Close-up view showing the side-chain arrangement of the residues Lys232, Pro233, Trp236, Trp244 and Pro246 in CRT(221-256), which are all affected by ring-current shifts due to proximity to the indole rings (Table 2). (c) Ribbon drawing of one of the 20 CRT(221-256) conformers shown in (a). The b-sheet is cyan, the a-helical turn is red, and the residues Lys232, Pro233, Trp236 and Trp244 of the hydrophobic cluster are shown in green as all-heavy-atom space-filling models.

PROCHECK

	Headers