Abstact

Gephyrin, the principal scaffolding protein of inhibitory postsynaptic densities, clusters glycine and GABA(A) receptors via multivalent interactions. It features structured N and C terminal domains connected by an intrinsically disordered linker. Although the structural and functional properties of its terminal domains are well characterized, the mechanism by which full-length gephyrin organizes into higher-order complexes remains unresolved. Here, we combine biochemical reconstitution, cryo-electron microscopy, and mutational analyses to elucidate the structural logic of gephyrin oligomerization. We demonstrate that gephyrin adopts a stable dimeric assembly which constitutes the basic unit for both linear and oblique tetramers as well as linear hexameric arrangements. High resolution structures reveal a critical segment of the flexible linker that adopts two distinct conformations, one of which occludes the receptor-binding site. This segment harbors key phosphorylation sites, suggesting a regulatory control mechanism. Our findings redefine the architecture of inhibitory postsynaptic sites and reconcile gephyrin oligomerization models with published in-situ postsynaptic densities characterized by cryo-electron tomography.