Extended Data Fig. 6|. Conformational changes at IL2 and IL3 interfaces correlate with interdomain twist angle.

a, The C loop contains residues S251 and D253, which interact with Y67 in the N domain in the inactive state of arrestin (cyan, left). In the rhodopsin-bound crystal structure, this network of residues separates when IL2 binds in the central crevice between the N and C domains (purple, right). b, c, We measured separation between the Y67–S251 side-chain hydroxyl oxygens (b) and between the C143–D253 Cα atoms (c). Conformational changes at the IL2 interface correlate with interdomain twist angles. This is particularly noticeable in simulations starting from the inactive state but with the arrestin C tail removed (green), where increased interdomain twist angles correlated with disruption of the Y67–S251 interaction (R² = 0.35) and with increased separation distance between the two domains, as measured through the C143–D253 Cα distance (R² = 0.36) (six independent simulations). Plots and correlations refer to trajectories downsampled every 10 ns, with no frames removed at the beginning of simulation. One caveat is that in simulations started from active state without the arrestin C tail, the interdomain crevice frequently collapsed at the beginning of simulation, so that even when arrestin visited more active interdomain twist angles, the crevice did not re-open. It is possible that these simulations reached a local energy minimum not typically visited in the equivalent simulations started from the inactive conformation. d, Conformational changes at the IL3 interface correlate with interdomain twist angles. Compared to the inactive state (blue) of arrestin, in the active state (purple), the back loop, located in the arrestin C domain (residues 311–320), extends away from the arrestin body (motion indicated by the black arrow). In this conformation, the back loop contacts the third intracellular loop in rhodopsin via an ionic interaction between R318 (arrestin) and E239 (rhodopsin). e, The position of the back loop correlates with the interdomain twist angle for simulations of arrestin with its C tail removed, starting from either the inactive (green) or active (purple) state (R² = 0.50 and R² = 0.58, respectively; six independent simulations). Back loop position is measured by projecting the coordinates of the back loop onto the vector connecting the crystallographic inactive- and active- gate back structures (see Methods). f Similarly, in simulations of arrestin bound to the receptor core only, movement away from active interdomain twist angles weakly correlated with disruption of the R318–E239 interaction (R² = −0.14; six independent simulations). Our simulations therefore indicate that interaction between arrestin and receptor at IL3 may control the interdomain twist angle. We speculate that this occurs because the back loop is coupled to the C loop via a set of β-strands. Thus, the receptor is likely to also modulate interdomain twisting by extending the shape of the back loop. When the back loop moves towards its active conformation, its motion appears to couple to the C domain through β-sheet formation with the C loop. Indeed, previous studies have indicated that acidic residues on IL3 might facilitate arrestin engagement. For example, an acidic residue on IL3 of the human luteinizing hormone receptor is critical for binding to arrestin-2 and arrestin-3, albeit to different extents for each⁶⁵. Our simulations support the idea that binding via the IL3 interface could help to trigger arrestin activation. Arrestins 1 to 4 share a conserved basic residue at position 313 (bovine arrestin-1 numbering). A qualitative examination of GPCR sequences reveals that several receptors, including the M2 muscarinic receptor, melatonin receptors, β2AR, A2AR, NTS1R, apelin receptor and H1R, all contain acidic residues at the 5×73–5×75 positions (GPCRdb numbering⁶⁶), which extend into ICL3 and may facilitate arrestin activation in the absence of R_P-tail phosphorylation.