(a) Chemical cross-linking of CRT(189–288) and ERp57. The homobifunctional cross-linker disuccinimidyl glutarate was added to solutions of CRT(189–288) alone (data not shown), ERp57 alone (lane 1), or an equimolar mixture of the two proteins (lane 2). After incubation for 30 min on ice, the reaction was quenched with an excess of glycine. Potential protein complexes were analyzed by 12% reducing SDS/PAGE, and the gel was stained with Coomassie blue. Molecular mass standards given in kDa are indicated on the left, along with the position of ERp57. The suggested position of the CRT(189–288)/ERp57 complex is indicated by *. (b) The interaction of CRT(189–288) and ERp57 studied by ELISA. Potential ligands for CRT(189–288) were coated in microtiter wells, and the interaction was probed with biotinylated CRT(189–288), followed by the binding of a primary antibody directed against biotin and a horseradish peroxidase-conjugated secondary antibody. Development by the addition of BM Blue (3,3′–5,5′–tetramethylbenzidine) (Roche Molecular Biochemicals) substrate gives rise to a signal at 450 nm for positive enzymatic reactions. BSA, calmodulin (CaM), and ubiquitin (Ubi) were used as control proteins. The data presented in the graph are mean values of triplicates. The experiment was repeated three times with closely similar results. Standard deviations are given by the vertical bars. Nonspecific binding of primary and secondary antibodies to wells coated with protein ligand in the absence of biotinylated CRT(189–288) was subtracted for each well. (c) Binding isotherm describing the formation of the CRT(189–288)/ERp57 complex followed by ITC at 8°C. ● represent the integrated heats at each injection after correction for nonspecific heat effects and normalization for the molar concentration. The continuous line visualizes a least-squares nonlinear fit of the data according to a 1:1 binding model defined by the following parameters: n = 1.04 ± 0.03, K_d = (9.1 ± 3.0) × 10⁻⁶ M [K_a = (1.1 ± 0.3) × 10⁵ M⁻¹], and ΔH = 2.1 ± 0.1 kcal mol⁻¹.

Visual display of the molecular regions of CRT(189–288) that are in direct contact with ERp57. (a) Ribbon representation. (b) Space-filling model displaying the molecular surface of the NMR structure. The residues for which the ¹⁵N–¹H-moieties show chemical shift changes caused by interactions with ERp57 in excess of 0.2 ppm along the ¹⁵N dimension are colored blue, those for which no cross peak is observed in the [¹⁵N,¹H]TROSY spectrum of the complex are red, and those for which the peak intensity relative to free CRT(189–288) is weaker than 3% are orange. In a the positions of selected residues areindicated.

Scheme for a possible mode of cooperative interaction between CRT and ERp57 in assisting the folding of a glycoprotein based on currently available structural, biochemical, and cell-biological data (2, 7, 8). The substrate glycoprotein (blue) binds to the CRT lectin domain (red) by means of a branched oligosaccharide. CRT and CNX both interact specifically with the Glc₁Man₉GlaNAc₂ form of the oligosaccharide (26–28). The interaction of the terminal glucose (light blue circle) with the binding site (white circle) would place the glycoprotein polypeptide chain in the partially solvent-shielded cavity bounded by the CRT lectin domain, the P-domain, and ERp57 (orange) bound to the distal end of the protruding P-domain. It is further hypothesized that the CRT-ERp57 interaction places the thiol-disulfide oxidoreductase favorably for the formation of intermolecular disulfide bonds (S—S) with the glycoprotein.

(a and b) ¹⁵N–¹H correlation NMR spectra used to delineate the surface areas of CRT(189–288) that interact with ERp57. These spectra contain peaks corresponding to backbone amide ¹⁵N–¹H groups and, in the lower left, indole ¹⁵N–¹H groups of tryptophyl residues. Color-coded peaks indicate outstandingly large effects from ERp57 interactions: Blue circles identify cross peaks with chemical shift differences of more than 0.2 ppm along the ¹⁵N dimension between free and ERp57-bound CRT(189–288). Red circles identify cross-peak positions in free CRT(189–288) for which no counterpart could be observed in the spectrum of the complex. The color-coded cross peaks are labeled with the sequence-specific resonance assignments. (a) [¹⁵N,¹H]TROSY spectrum of free ¹⁵N,²H-labeled CRT(189–288) (protein concentration = 0.4 mM, polarization transfer time = 5.4 ms, data size 200 × 1,024 complex points, zero-filling to 512 × 2,048 points, t_1,max = 88 ms, t₂,_max = 98 ms, total measuring time 2 h). (b) [¹⁵N,¹H]TROSY spectrum of a solution of ¹⁵N,²H-labeled CRT(189–288) and unlabeled ERp57 [protein concentrations of 0.14 mM for ¹⁵N,²H-labeled CRT(189–288) and 0.38 mM for ERp57, polarization transfer time = 3.4 ms, data size 100 × 1,024 complex points, zero-filling to 512 × 2,048 points, t₁,_max = 44 ms, t₂,_max = 98 ms, total measuring time 24 h]. (c) Plot of the relative peak intensities, I_rel, of the [¹⁵N,¹H]TROSY cross peaks in the CRT(189–288)/ERp57 complex and free CRT(189–288) versus the amino acid sequence of CRT(189–288). The Trp indole cross peaks are listed separately on the right. The black horizontal bar spanning the polypeptide segment 225–251 indicates that for these residues the cross peaks in the complex have either large chemical shift differences (> 0.2 ppm along the ¹⁵N dimension) or very small signal intensities [< 3% of the intensity in free CRT(189–288)] (see text). P designates proline residues, NA stands for residues for which no resonances could be assigned, and * indicates residues for which no reliable data could be measured because of spectral overlap.

Titration of the ¹⁵N,²H-labeled CRT(189–288)/ERp57 complex with unlabeled CRT(189–288). (a-c), Cross sections from [¹⁵N,¹H]TROSY spectra along the ω₁(¹⁵N) dimension for the residues A230, K232, and I249 of ¹⁵N,²H-labeled CRT(189–288). The numbers on the left of each trace refer to the solution conditions given in Table 1. (d) Cross sections from [¹⁵N,¹H]TROSY spectra along ω₂(¹H) for residue E234. (e) K_d determination based on the titration data for residue K232. ■ represent the experimental ¹⁵N chemical shifts of K232 at discrete values of the ERp57/CRT(189–288) molar ratio (Table 1). The curve visualizes the fit from which the ¹⁵N chemical shift of the CRT(189–288)/ERp57 complex [δ(¹⁵N_complex)] was determined, using the relation δ(¹⁵N) = δ(¹⁵N_free) + [δ(¹⁵N_complex) − δ(¹⁵N_free)] ([ERp57]/[CRT(189–288)])/(([ERp57]/[CRT(189–288)]) + K_ratio) where δ is the chemical shift and K_ratio the K_d expressed as molar ratio, and the brackets indicate molar concentrations of the respective compounds. Subsequently, the K_d value was calculated at each experimental point by using the relation K_d = [CRT(189–288)][ERp57]/[CRT(189–288)/ERp57], yielding an average value of K_d = (18 ± 5) × 10⁻⁶ M.