Fig. 2: Sequence and structural basis of ligand recognition by evolved PYR1 sensors.

a, Sequence diversity of cannabinoid receptor ligand-binding pocket residues (mutant residues are shown in bold type). The minimal ligand concentrations required for Y2H signal generation are indicated at right (Supplementary Fig. 2 shows full data, including mutations outside the pocket). The heatmap shows the ligands screened clustered by their pairwise Tanimoto distance scores calculated using ChemMine³³; blue indicates high similarity, and orange has lower similarity. b, Representative optimized sensor Y2H β-galactosidase responses to the ligands indicated; PYR1^CBDA was evolved for recognition of CBDA, PYR1^CP for CP 47,497, PYR1^4F for 4F-MDMB and PYR1^WIN for WIN 55,212-2. c–e, Structural basis for cannabinoid recognition. c, WIN is colored yellow, and key ligand-contacting residues are indicated with dashes. The Trp-lock water network that stabilizes binding is shown at top. d, Relief of steric clash by the evolved receptor. e, Structural poses of WIN in PYL2-bound (top) and CB2-bound (bottom, 6PT0) structures.