Multimodal binding of collybistin controls gephyrin filament formation in synaptic clustering

The multifunctional protein gephyrin clusters glycine and GABA type A receptors at inhibitory postsynapses by oligomerization of its G- and E-domain. Collybistin, a gephyrin-interacting nucleotide exchange factor, mediates gephyrin recruitment and clustering at specific GABAergic postsynapses via phosphoinositide interaction. Using single-particle cryo-electron microscopy, we uncovered the structural basis of collybistin-regulated gephyrin clustering, which controls gephyrin oligomerization in two ways: While collybistin inhibited E-domain dimerization-dependent filament formation of gephyrin, the presence of phosphoinositides promoted the formation of stable gephyrin-collybistin complexes that underwent E-domain dimerization-dependent filament assembly. Within these complexes, collybistin binds at distinct positions, with different stoichiometries and conformations, either promoting or restricting filament assembly and thus regulating filament formation and elongation. Our study highlights the critical role of E-domain dimerization-dependent filament formation for postsynaptic clustering and demonstrates its tight regulation by collybistin, driving specific oligomerization at phosphoinositide-enriched GABAergic synapses.