Figure 4: Impact of the phosphorylation pattern on β-arrestin 2 conformational dynamics.

(A) Heat scatter plots of the interdomain rotation angle of β-arrestin 2 (a measure of activation) and probe distances (probe 1 to 5) to the RLuc domain. The interdomain rotation angles and corresponding probe distances have been computed for simulation frames sampling β-arrestin 2 inactivation. A structural model of the construct used in the FlAsH BRET conformational assay (N-domain in grey and the C-domain in blue) highlights the positions of probes 1-5 (red spheres) and the N-terminal end (residue 8 in our model) used as an approximation for the RLuc attachment at the N-terminal end of β-arrestin 2 (green spheres), as the Rluc domain is absent in the simulated system. (B) Binding mode of the CXCR3 WT C-tail to β-arrestin 2. Negatively charged residues (Asp, Glu or phosphorylated Ser and Thr) on the C-tail are depicted in licorice and their Cα atoms are highlighted with red spheres. Positions mutated within this study are labeled. The inset provides a detailed depiction of the lariat loop region of β-arrestin 2 (blue) and interactions with negatively charged residues of the C-tail. Bar charts demonstrate the stability of polar interactions between K294 of the lariat loop and S358 and E359 of the C-tail during MD simulations. In the bar plots, systems with a low FlasH probe 4-5 resonance energy transfer (RET) are colored in red (WT and T360AS361A). System with an intermediate or high FlasH probe 4-5 RET are colored in black (4xA, S355A/S356A, L344X). (C) Distribution plots of the interdomain rotation angles of β-arrestin 2 in complex with C-tail mutants. The distribution of the interdomain rotation angles have been computed over the accumulated simulation frames for β-arrestin 2 in complex with C-tail of the WT versus mutants. The peaks of the plots indicate the most explored conformations for the WT system (WT_peak) versus mutant systems (e.g. L344X_peak). Systems are grouped based on probe 4-5 RET. Intermediate and high probe 4-5 RET goes along with a shift of the distribution peak to lower rotation angles compared to the WT system (low probe 4-5 RET).