Fig. 4. (A) Part of 3D X-ray crystal structure of HA top (PDB ID: 2VIR; Fleury et al. 1998)13 with the residues of the peptide (C76–F87) highlighted with sticks. A disulfide bond in HA top (C64–C76) provides C76 (highlighted in orange) as the N-terminal amino-acid of the model peptide. The residue bearing the mannosylation (D/N81, or mannosylated residue in the model peptide) is shown and labelled in purple. (B) STD-NMR of CVN2L0-peptide complex: upper trace: 1H NMR spectrum of the native peptide P2 (DM), *(asterisk) marked the appr. shift range of saccharides. Middle trace: STD-NMR spectrum of the free peptide (negative control experiment). Lower trace: STD-NMR spectrum of DM in the presence of CVN2L0 indicating complex formation at 3 ppm. a.u. = arbitrary units. (C) STD-NMR of CVN2L0-V3 and DM. (D) STD-NMR of CVN2L0-V3 and peptide P3 (MM). STD in the difference spectrum related to peptide alkyl-resides (δ1H = 1 ppm) and its mannosylation site (δ1H = 3–4 ppm). Chemical exchange effects arise due to lower affinity with MM upon site-specific recognition of D81X-E82-T83 (X = mannosylated glycine-derivative). CVN2L0-V3 (C58W and C73M in both high-affinity carbohydrate binding sites). 100 μM peptide was measured over 10 μM protein. Saturation was performed with a Gaussian pulse train lasting for 4 s at −1.0 ppm (1000–4000 scans were needed). Water suppression was achieved with a double Watergate echo (excitation sculpting).
