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

Ortiz-Lopez, D.; Hove, T. T.; Huhn, C.; van gen Hassend, P. M.; Sander, B.; Campbell, B. F. N.; Tyagarajan, S. K.; Plueckthun, A.; Maric, H. M.; Boettcher, B. M.; Schindelin, H.

doi:10.1101/2025.09.01.673457

bioRxiv · 10.1101/2025.09.01.673457

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

Ortiz-Lopez, D.·Hove, T. T.·Huhn, C.·van gen Hassend, P. M.·Sander, B.·Campbell, B. F. N.·Tyagarajan, S. K.·Plueckthun, A.·Maric, H. M.·Boettcher, B. M.·Schindelin, H.

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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.

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Ortiz-Lopez, D., Hove, T. T., Huhn, C., van gen Hassend, P. M., Sander, B., Campbell, B. F. N., Tyagarajan, S. K., Plueckthun, A., Maric, H. M., Boettcher, B. M., Schindelin, H.. 2025-09-04. Cryo-EM Structures of Higher Order Gephyrin OligomersReveal Principles of Inhibitory Postsynaptic Scaffold Organization. https://doi.org/10.1101/2025.09.01.673457

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