Figure 7.

Schematic representation of gephyrin-mediated CB opening and complex assembly. (a) Cartoon and surface representation of full-length CB (PDB: 4mt6) in its auto-inhibited conformation depicting the individual domains (SH3; light gray with white surface, DH; black with dark gray surface, and PH; gray with light gray surface). The figure illustrates the ensemble of sensors used in this study with the FlAsH attachment sites after amino acid residues 1 (F1_DA), 28 (F28_DA), 73 (F73_DA), and 99 (F99_DA) represented as green spheres, whereas CFP (cyan) was inserted after residue 456. Addition of gephyrin shifts the equilibrium toward an open state. In the closed, high-FRET state, the C-terminally attached CFP exhibits significant quenching (cyan), whereas the low-FRET, open state (CFP in teal) is characterized by reduced CFP quenching. (b) Cartoon representing the active-state double-mutant CB FRET sensor (F1_dmDA, the mutated residues are indicated by white dots), which is already in an open state, and its conformational change upon interaction with gephyrin.