Fig. 7. Different tICA macrostates are responsible for different biased signaling.

A Macrostates 1 (green), 5 (salmon), 6 (gray), and 7 (purple) with their corresponding specific micro-switches are shown in cartoon. Involved residues are shown in sticks. Because of the obvious fluctuation of the distance between N^3.35 and D^2.50 in representative trajectories of macrostates, the distance is shown in mean ± standard deviation form for all snapshots from the trajectories. AngII-induced Gq activation (B), β-arrestin 2 recruitment (C), Gi activation (D), and G12 activation (E) in HEK293 cells transfected with WT AT₁ receptor (black) or D^2.50N (green), N^3.35A (red), and Y^7.53I (yellow) mutants. Three independent experiments were performed and representative dose-response curves were shown. The bars indicate the mean ± SEM values. F Heat map of AT₁ receptor signaling signatures of WT AT₁ receptor and mutants. Blue and red squares represent decreased or increased activity compared with WT AT₁ receptor. The relative activities Δlog(τ/K_A) of WT AT₁ receptor and mutants in each signaling pathway calculated in (Supplementary Note 17) were expressed as a heat map. Color scales of the map are shown on the right. ΔBRET: the change of bioluminescence resonance energy transfer value.