Abstract
Little is known about how chaperones in the endoplasmic reticulum are organized into complexes to assist in the proper folding of secreted proteins. One notable exception is the complex of ERp57 and calnexin that functions as part the calnexin cycle to direct disulfide bond formation in N-glycoproteins. Here, we report three new complexes composed of the peptidyl prolyl cis-trans-isomerase cyclophilin B and any of the lectin chaperones: calnexin, calreticulin, or calmegin. The 1.7 Å crystal structure of cyclophilin with the proline-rich P-domain of calmegin reveals that binding is mediated by the same surface that binds ERp57. We used NMR titrations and mutagenesis to measure low micromolar binding of cyclophilin to all three lectin chaperones and identify essential interfacial residues. The immunosuppressant cyclosporin A did not affect complex formation, confirming the functional independence of the P-domain binding and proline isomerization sites of cyclophilin. Our results reveal the P-domain functions as a unique protein-protein interaction domain and implicate a peptidyl prolyl isomerase as a new element in the calnexin cycle.
Highlights
Calreticulin (CRT) [2] and a complex of GRP94, ERp72, BiP, CRT, and cyclophilin B [3]
Work from a number of groups, including our own, has shown that these processes are linked through the calnexin cycle wherein the trimming of the glycan of N-linked glycoproteins is tied to the folded state of the protein
Recruitment of the heterodimeric chaperone complex CNXERp57 is in turn regulated by the presence of a terminal glucose residue on the N-glycan
Summary
Protein Expression, Preparation, and Purification—Human cyclophilin B (residues 2–184 of the mature protein) was cloned into a pGEX-6P-1 vector (GE Healthcare, Uppsala, Sweden) and expressed in Escherichia coli BL21(DE3) in rich (LB) medium as a fusion with N-terminal GST tag. The crystals of unliganded cyclophilin B were obtained by equilibrating a 0.6-l drop of the cyclophilin B (7 mg/ml) in buffer (20 mM MES (pH 6.5), 50 mM NaCl), mixed with 0.6 l of reservoir solution containing 25% (w/v) PEG 1500, and 0.1 M MMT buffer (pH 6.0) and suspended over 0.6 ml of reservoir solution. The best cyclophilin1⁄7P-domain complex crystals were obtained by equilibrating a 1-l drop of the cyclophilin1⁄7CMG (residues 317–350) mixture in a 1:3 molar ratio (8 mg/ml) in buffer (20 mM MES (pH 6.5), 50 mM NaCl), mixed with 1 l of reservoir solution containing 22% (w/v) PEG 8000, 10 mM ZnCl2, and 0.1 M Tris buffer (pH 7.0) and suspended over 1 ml of reservoir solution. NMR spectra were processed beads, cyclophilin B was able to retain CRT to the same extent using NMRPipe and analyzed with XEASY
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