Gephyrin filaments represent the molecular basis of inhibitory postsynaptic densities

The multifunctional protein gephyrin clusters glycine and GABA type A receptors at the postsynaptic membrane in the CNS. Gephyrin has been proposed to form the inhibitory postsynaptic density by liquid-liquid phase separation, involving a complex interplay between receptor binding and oligomerization via its conserved G- and E-domains. Our single particle cryo-EM analyses uncovered that a single receptor-loop can occupy simultaneously both previously identified receptor-binding sites within gephyrin E-domain dimers. More importantly, dimerization allowed the formation of filaments in which two Gephyrin E-domains are linked by Z-shaped interfaces formed by two subdomain two (SDII) of adjacent dimers. Deletion of SDII abolished the formation of filaments, phase separation and synaptic receptor clustering. Additional structural data rationalizes two mutations previously identified in human patients with epileptic encephalopathy, thus underscoring the functional importance of the identified molecular mechanism for postsynaptic density formation by gephyrin.