Fig. 4.

Ligand–receptor interaction maps and sequence profile-guided design enable the isolation of high-affinity sensors. (A) Selected chemical structures of coumarin hits used to generate the sequence profile of the coumarin-target library: osthole (1), imperatorin (2), and angelicin (3). Receptors for 4-methylumbelliferone (4) and 7-methoxycoumarin (5) were not identified in the initial screen; sensors for these compounds were isolated from the coumarin-target library. (B) Sequence profile of the coumarin targeted library and binding pocket mutations of PYR1 receptors activated by compounds (1–5). (C) Yeast-hybrid β-galactosidase colony assays comparing PYR1 sensor response of the initial sensor and high-affinity sensors isolated from the coumarin-targeted library. The targeted library yielded PYR1 receptors for (4 and 5), which were not identified in the initial data generation screen. The minimum dose–responses are shown in comparison to the mock condition. SI Appendix, Fig. S2 shows additional growth and reporter dose–response assays for the coumarin sensors. (D) Chemical structures of selected ligands used to design the sequence profile of a TNT target library: 2,4-dinitrophenol (6), chloroxylenol (7), 2,4,6-trihydroxybenzoic acid (8), 2,6-dihydroxybenzoic acid (9). Hits for trinitrotoluene (TNT; 10), dinitrotoluene (DNT; 11), and 2-amino-dinitrotoluene (2ADNT; 12) were not identified using the screening libraries. Sensors for these were isolated from the TNT target library. (E) Sequence profile of the TNT target library (TNTv2) and binding pocket mutations for TNT, DNT, and 2ADNT receptors isolated from the library. The sequence profile used for library construction additionally includes 117L/W and 160A/V (Dataset S4). (F) Yeast-hybrid β-galactosidase colony and cell-based titration assays for PYR1^TNT, PYR1^DNT, and PYR1^2ADNT for their on-target ligands. Triplicate data points are shown. The inset data indicate the EC₅₀ and range.