Fig. 5
From: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers
Kinase-mediated ligand-free dimers adopt two ECM dimer architectures. a FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells treated with 1 μM erlotinib, compiled from 29 FLImP measurements (CI ≤ 6 nm), decomposed into a sum of four components. The concentration of CF640R-Affibody was 4 nM. b Number of measurements consistent with the mean distances resolved in the FLImP distribution of wtEGFR (Fig. 2b) (associated FLImP distributions in Supplementary Fig. 13). Errors were assesed with bootstrap-resampling12. c wtEGFR and IIIV/KKRE-EGFR phosphorylation in Y1173 on CHO cells treated or untreated with 10 mM MβCD. Box plots show inclusive median as a line, 25th and 75th quartile as edges, calculated on n = 3 repeats (representative western blots in Supplementary Fig. 14). d As a treated with 10 mM MβCD, from 20 FLImP measurements (CI ≤ 7 nm). e Pairwise particle colocalisation fraction from two-colour SPT on live cells at 37 °C. f Duration of pairwise interactions (τON) in e. Horizontal spreads separate data points (~5000) within each condition. g Crystal structure of tethered wtEGFR (PDB ID 1NQL5) highlighting the location of I545K, I556K, I562R, and V592E (yellow). Colours: DI (green), DII (red), DIII (blue), DIV (grey), EGF (orange). h Head-to-head dimer highlighting the residues mutated in the IIIV/KKRE mutant (yellow). i As a but for the IIIV/KKRE-EGFR mutant from 22 FLImP measurements with CI ≤ 7.5 nm. j FRET-DOCA from DI (blue) and DIII (red) to the membrane for wtEGFR + erlotinib, and L680N-EGFR, derived from measurements in Supplementary Fig. 10. FRET probes as in Fig. 4e. k Ratio between CF640R-9G8-NB and Alexa 488-EgB4-NB binding after chemical fixation. wtEGFR, blue; wtEGFR + erlotinib, green; L680N-EGFR, red. Line, median; box edges, 25th and 75th quartile, crosses 5th and 95th quartile, calculated over 30 repeats. Example images and analysis are in Supplementary Fig. 15. l As a but for L680N-EGFR-expressing cells, from 20 FLImP measurements (CI ≤ 6 nm). Lower resolution (8 nm) versions of a, d, and l with ~2-fold more FLImP measurements show the profile of the distributions is conserved (Supplementary Fig. 16)
