Cryo-EM structures of eight IMiDs in full CRL4CRBN ubiquitylation complexes reveal that next-generation IMiDs achieve enhanced therapeutic efficacy by simultaneously engaging both the canonical CRBN TBD pocket and a cryptic gluing-driven interfacial (GDI) pocket formed by the IKZF3 non-degron ZF3 motif, providing a structural basis for rational design of improved molecular glue degraders with enhanced potency and selectivity.

Immunomodulatory imide drugs (IMiDs) redirect the CUL4–RBX1–DDB1–CRBN (CRL4CRBN) ligase to ubiquitylate and degrade disease-linked proteins, but the full picture of how IMiDs glue the neosubstrate within the whole CRL4CRBN complex remains unknown. We determined cryo-electron microscopy structures of eight approved or clinical IMiDs in full CRL4CRBN ubiquitylation complexes at resolutions up to 3.4 Å, and revealed how these structurally distinct IMiDs exploit the structural plasticity of CRL4CRBN to organize conformationally conserved and compact active ubiquitylation assemblies with the neosubstrate IKZF3. Four “next-generation” IMiDs were found to additionally engage a cryptic gluing-driven interfacial (GDI) pocket at the non-degron zinc finger 3 (ZF3) of IKZF3, which contributes to their enhanced efficacy and neosubstrate specificity. The identification of cryptic gluing-driven pockets formed only in full ubiquitylation complexes provides new structure-directed design opportunities for IMiDs to improve therapeutic efficacy.