Fig. 1. α5 subunit engineering and structure validation.

a, Schematic top-down view of the subunit make-up of the full heteromeric α1β2/3γ2 receptor versus the engineered α5V1, α5V2 and α5V3. The homomer site is created between residues from the α5 principal face (red) and substituted γ2 residues introduced into the complementary face (yellow). Asterisk indicates site is occupied by drug in structure. b, Chemical structures of flumazenil (FLZ), bretazenil (BRZ) and diazepam (DZP), with benzodiazepine ring system colored red. c–f, Structural model ribbon representations of drug binding modes for (c), FLZ bound to α5V1 (d), BRZ bound to α5V2 homomeric site (e), BRZ bound to α5V2 heteromeric site (f), DZP bound to α5V3 site, showing the α5V α5 principal face (red) and γ2 substituted complementary face (yellow). Bound drugs shown as sticks: oxygen, red; nitrogen, blue; fluorine, cyan; chlorine, green; bromine, brown. Superposed previously solved α1β2γ2 structure bound by flumazenil (PDB 6X3U) or α1β3γ2 bound by diazepam (PDB 6HUP) are shown in white. Loop-C, which binds over the pocket, like a cap, is not shown for clarity. For reference, equivalent complementary face residue numbering of α5V3 Y49, A70 and T133, in wild-type γ2 is Y58, A79 and T142, respectively.