Journal of Molecular Biology
NMR Structures of 36 and 73-residue Fragments of the Calreticulin P-domain
Introduction
In the endoplasmic reticulum (ER), two homologous lectin chaperones, calnexin (CNX) and calreticulin (CRT), assist the folding and quality control of proteins carrying N-linked glycans.1 Whereas CNX is membrane-bound, CRT is a soluble lumenal protein. Both proteins interact specifically with glycoproteins carrying monoglucosylated (Glc1Man7–9GlcNAc2) trimming intermediates of the triglucosylated core glycan (Glc3Man7–9GlcNAc2).2., 3., 4. Release from CNX and CRT is ensured by glucosidase II, which removes the remaining glucose from the glycan. Once the glycoprotein has adopted its native structure upon release, it is free to leave the ER and proceed along the secretory pathway. Alternatively, if it is still not correctly folded, the glycoprotein is recognized by the UDP-Glc:glycoprotein glucosyltransferase, which thus serves as a folding sensor.5 By re-adding a glucose unit to the glycan, this enzyme promotes renewed association with CNX and CRT.
Both chaperones also cooperate with the thiol-disulfide oxidoreductase ERp57, which is a close homolog of the protein disulfide isomerase (PDI).6., 7. Like PDI, ERp57 contains four thioredoxin-like domains with active site –CXXC– sequence motifs located in the N and C-terminal domains. In vivo, ERp57 promotes disulfide bond formation in glycoprotein substrates bound by CNX and CRT through the formation of transient intermolecular disulfide bonds.8 Overall, the CNX/CRT chaperone system increases folding efficiency, and prevents aggregation and premature ER exit of newly synthesized glycoproteins.
Both CNX and CRT contain a central proline-rich region, the “P-domain”. It consists of two types of sequence repeats with 17 residues (type 1) and 14 residues (type 2), respectively. CNX contains four repeats of each type arranged in a 11112222 fashion, whereas CRT contains three repeats of each type in a 111222 arrangement (Figure 1(a)). The three-dimensional structures of the CNX and CRT P-domains are highly similar, except for the presence of the additional pair of sequence repeats in CNX.9., 10. The CRT P-domain comprising residues 189–288, CRT(189–288), forms an extended hairpin fold with the N and C termini in close proximity (Figure 1(b)).10 The structure is stabilized by three short antiparallel β-sheets and three small hydrophobic clusters, each involving two tryptophyl rings packed against the aliphatic side-chains of a prolyl and a lysyl residue. The presence of the three equidistantly spaced β-sheets and the three hydrophobic clusters clearly reflects the threefold repetition of sequence repeats in CRT(189–288) (Figure 1).
Recently, the crystal structure of the CNX ectodomain, which includes the P-domain, was solved.9 It is characterized by a compact lectin domain showing a β-sandwich structure, from which the elongated P-domain extends. Based on the close sequence similarity between the two proteins, the global three-dimensional structure of CRT can be assumed to be similar to that of CNX. In the linear sequence of the two proteins, the P-domain is inserted in-between the two peripheral regions, which are in close proximity in the three-dimensional structure and form the β-sandwich and a structural Ca2+-binding site.
The role of the P-domain in glycoprotein folding has become clearer with our recent finding that the CRT P-domain interacts with the co-chaperone ERp57.11 Specifically, we were able to show that the interaction with ERp57 occurs through residues 225–251 at the tip of the P-domain structure.11 Thus, in the binary CRT–ERp57 complex, the lectin domain, the P-domain and ERp57 appear to form a partially solvent-shielded “reaction chamber”, where folding of the bound glycoprotein could take place while access for other folding intermediates and chaperones is restricted. In addition, the positioning of ERp57 at the tip of the P-domain might facilitate the interaction with cysteine residues in the substrate glycoprotein.
Here, we describe the structure determination by solution NMR spectroscopy of two polypeptide fragments from the CRT P-domain, CRT(221–256) and CRT(189–261), consisting of 36 and 73 residues, respectively. The results indicate that CRT(221–256) is a promising candidate for use in further studies into the nature of the interaction between CRT and ERp57, since it comprises the region of CRT that is involved in the direct contacts with ERp57,11 and is demonstrated to interact with ERp57 with comparable affinity as the intact CRT P-domain.
Section snippets
Design, expression and physical–chemical characterization of the CRT P-domain fragments CRT(221–256) and CRT(189–261)
To identify potential independently folding fragments of the CRT P-domain, limited proteolysis experiments were carried out on a CRT construct comprising residues 189–300. A subtilisin-resistant fragment comprising residues 189–261 was identified by mass spectroscopy and N-terminal sequencing (K. Ng, J. Peterson, W. Weis & A.H., unpublished data). As shown in Figure 1, this fragment encompasses the three type 1 repeats and approximately the first one and a half type 2 repeats. In the structure
Discussion
The present study shows that the unusual hairpin-type fold of the 100-residue CRT P-domain is maintained in two smaller fragments of this polypeptide chain, containing 73 and 36 residues, respectively. The fact that the larger subdomain, CRT(189–261), contains an N-terminal disordered tail of residues 189–213 (Figure 4, Figure 6), further illustrates that the formation of this fold-type depends critically on inter-strand contacts in the hairpin, since the partner strand segment for residues
Rapid autonomous fragment test (RAFT) calculation
As described in detail by Fischer & Marqusee,12 the RAFT algorithm first identifies all inter-residue contacts present within a protein of known three-dimensional structure. Next, continuous segments are scored based on the number of internal contacts within a given segment as compared to the number of external contacts. Finally, the score is normalized to the length of the segment. For the present calculation, the CRT(189–288) structure was used as input. The shortest fragment identified by
Acknowledgements
We thank Dr R. Riek for advice on the resonance assignment procedures used for CRT(189–261), Dr K. F. Fischer for help with the RAFT calculations, Dr M. Bouvier for critical reading of the manuscript, Eva Frickel for helpful discussions and preparation of ERp57, and Christiane Schirra for excellent technical assistance. Financial support by the Schweizerischer Nationalfonds (projects 31.51054.97 (A.H.) and 31.49047.96 (K.W.)) and the use of the computing facilities of the Competence Center for
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Present address: P. Güntert, RIKEN Genomic Sciences Center, W505, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan.