Cryo-EM Structures of Higher Order Gephyrin OligomersReveal Principles of Inhibitory Postsynaptic Scaffold Organization

Abstract

Gephyrin is the principal scaffolding protein of inhibitory postsynaptic densities, clustering glycine and GABAA 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, providing a mechanistic link between structural conformation and regulatory control. Our findings redefine the architecture of inhibitory synapses and reconcile gephyrin oligomerization models with published in-situ post-synaptic densities characterized by cryo-electron tomography.