Fig. 4. Arrestin-biased, balanced, and G-protein-biased ligands favor distinct binding-pocket conformations.

(A) Structures of representative β-arrestin-biased, balanced, and Gq-biased AT₁R ligands. All ligands studied in this work are shown in Fig. S1. (B) In comparison to β-arrestin-biased ligands, AngII drives L3.36 of the binding pocket toward its TM2-proximal position, which favors the canonical active conformation, and Gq-biased ligands do so even more (P = 0.002 for β-arrestin-biased vs. Gq-biased ligands; see Methods) (left; the bar plot shows means and standard errors across 5 independent 2-μs simulations per ligand). When the F8 residue of AngII and Gq-biased ligands adopts a horizontal orientation, it pushes L3.36 toward the TM2-proximal position (right; gray and pink dashed lines indicate values for TRV023- and AngII-bound structures, respectively; traces are for a simulation with AngII bound, and additional simulation traces are shown in Fig. S6). RMSD is root-mean-square deviation (see Methods). (C) AngII and β-arrestin-biased ligands stabilize more inward TM6 positions in the binding pocket compared to positions favored by Gq-biased ligands (P = 0.005 for Gq-biased ligands vs. AngII; P = 0.001 for Gq-biased ligands vs. β-arrestin-biased ligands), as shown by the box plot at left (boxes extend from 25^th to 75^th percentile of simulation frames), because the ligand R2 residue interacts with D6.58 and D7.32 (right; dashed lines correspond to distance plotted at left). The more outward position of the extracellular portion of TM6 observed for Gq-biased ligands allows their F8 residue to adopt a horizontal orientation more frequently than that of AngII (see also Fig. S7).