Fig. 3

Epitope mapping by ¹⁵N NMR shows that all VHHs bind near the C terminus of Smt3p. (A–C) ¹H ¹⁵N-TROSY-HSQC spectrum of ¹⁵N-labeled Smt3p in the presence of excess VHH1_SMT3 (red) (A), VHH2_SMT3 (blue) (B), and VHH4_SMT3 (pink) (C). The spectra are overlaid on those of Smt3p in the absence of added VHH (black). Individual Smt3p residues that show estimated lower limit for combined chemical shift changes in parts per million (ppm) upon VHH binding are shown in the middle panels. Residues with chemical shift changes with ppm > 0.1 are indicated in yellow, and those with ppm > 0.2 are indicated in red. Residues with > 0.1 ppm, yellow and red, mapped onto a surface rendering of Smt3p (PDB:1EUV) are shown on the right. (D) Comparison of ¹H ¹⁵N-TROSY-HSQC spectrum (left) and estimated lower limit of combined chemical shift changes of Smt3p residues (right) in the presence of VHH2_SMT3 (blue) overlaid on the spectrum obtained in the presence of VHH4_SMT3 (pink) highlights the similarity of the presumptive contact residues. (E) Overlay of the spectra collected in the presence of VHH1_SMT3 (red) and VHH2_SMT3 (blue) showcases the differences in residues affected by their respective VHH binding. The inclusion of VHH1_SMT3 shows stronger perturbation towards the C-terminus of Smt3p consistent with a distinct mode of binding. Note that the K_d values for VHH1_SMT3 and VHH2_SMT3 are similar (Table 1).