Fig. 4: Click labeling of BCNK at different locations on the protein surface.

a Site-specific signatures of click efficiency (CE) in the absence and presence of IL-4 measured under identical labeling conditions. Domain-specific averages (neutralizing sites omitted) are slightly reduced in the presence of IL-4 (dashed lines). The range of two standard deviations (shaded, light gray) was calculated for each domain separately. Significance was tested with two-sided Welch’s t-test. Asterisks indicate p < 0.05 (*; K22B: p = 0.019), p < 0.01 (**; S44B: p = 0.0070, E189B: p = 0.0095), p < 0.001 (***; S30B: p = 0.0006, K87B: p < 0.0001, K94B: p < 0.0001, K97B: p < 0.0001, T113B: p = 0.0002, E141B: p <  0.0001). Box-and-whisker plots indicate first and third quartile (box), median (horizontal line), and 1.5 times the interquartile range (whiskers). Descriptive statistics containing the number of cells measured for each condition and the number of biological replicates are listed in Supplementary Table 5. b B-factors of atoms forming the BNCK peptide bonds from MD simulation of IL-4Rα (PDB: 3BPN) show reduced motion at key positions of IL-4 ligand binding. c Scheme of the terminal catchbox with dimensions resembling the tetrazine moiety (13.5 Å x 4.5 Å x 3.5 Å) used for sampling atoms during MD simulations. d Average structures from 250 ns MD trajectories of receptor mutants (K22B, E94B). Atoms entering the catchbox along the trajectory are highlighted (‘spheres’ representation, color code indicates the frequency of occurrence). Despite similar CE, the contact area for K22B is widely distributed, whereas in E94B the terminal ring populates a specific hydrophobic hotspot (zoom-in). e Definition of polar and azimuthal angles of the terminal BCNK ring as a measure for rotor flexibility. f Probability distribution of polar and azimuthal angles of the BCNK ring for the receptor mutants (K22B, E94B) corresponding to (d). Source data are provided as a Source Data file.