van gen Hassend, P. M. | Search bioRxiv

bioRxiv2025

A Structural Code for Assembly Specificity in GID/CTLH-Type E3 Ligases

GID/CTLH-type E3 ligases assemble into conserved ring-shaped architectures built from repeating LisH-CTLH-CRA modules, yet the molecular rules that enforce their highly specific subunit arrangement have remained unknown. Here, we decode the structural "assembly specificity code" that governs CRA-CRA pairing. Using crystal structures of multiple CTLH-CRA domains, including the RanBP9-muskelin heterodimer, integrated with quantitative binding analyses, we show that several interfaces operate with exceptionally high affinity, reaching the picomolar range, and that conserved sequence and geometric features enable each subunit to only select cognate partners. Strikingly, targeted perturbations of these features are sufficient to reprogram pairing preferences, enabling engineered subunits such as RanBP10 or Twa1 to adopt non-native interaction partners. These findings reveal the molecular logic that preserves the architecture of GID/CTLH-type E3 ligases and demonstrate that their assembly code is both decipherable and engineerable, providing a conceptual foundation for reconfiguring these ring-shaped E3 ligases.

biochemistry↗

bioRxiv2025

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

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.

neuroscience↗