Extension of the GlyRβ binding site on gephyrin. Extension of the GlyRβ binding site on gephyrin. A, EDC-based crosslinking of βL-wt (378–426) and gephyrin. Gephyrin (Geph) and GlyR β-loop (βL) were treated (lanes 2 and 3) or not treated (lane 1) with EDC and separated by 6% SDS-PAGE (Coomassie staining). B, Identification of crosslinked peptides. Bands corresponding to the protein complexes shown in A were extracted and analyzed by peptide mass fingerprinting. The crosslinked peptides of gephyrin (purple) and the β-loop (green, red) were identified several times. C, Amino acid sequence of βL-wt with the localization of N- (green, 378–393) and C-terminal (blue, 414–426) flanking sequences of the core gephyrin-binding site (red, 394–426). Schematic representation of gephyrin domains with highlighted positions of identified peptides in the C- and E-domain (purple). D, Surface representation of a modeled trimeric full-length gephyrin (modified from (Belaidi and Schwarz, 2013) with highlighted peptides identified in the crosslinked gephyrin-GlyR β-loop complex. Dashed lines indicate regions in the βL for which structural information is lacking. Gephyrin protomer II and III: light gray; protomer I: E-domain, orange and G-domain, light orange; βL core sequence: red; βL N-terminal flanking sequence: green; βL C-terminal flanking sequence: blue; and crosslinked peptides of the gephyrin E-domain: purple. Nora Grünewald et al. eNeuro 2018;5:ENEURO.0042-17.2018 ©2018 by Society for Neuroscience