Cryo‐ EM reveals an autoinhibited dimeric state of the neddylated CRL2 FEM1B E3 ubiquitin ligase

Abstract Cullin–RING E3 ubiquitin ligases (CRLs) constitute the largest family of E3 ligases in eukaryotes and are estimated to mediate the ubiquitylation of approximately 20% of proteins degraded through the ubiquitin–proteasome system. CRL activity is generally stimulated by covalent modification of the cullin scaffold with NEDD8. Here, using cryo‐electron microscopy combined with biochemical and cellular ubiquitylation assays, we characterize an autoinhibited state of neddylated CRL2 FEM1B . Structures of N8‐CRL2 FEM1B in the absence and presence of the FNIP1 degron reveal a crown‐shaped homodimer stabilized by interprotomer CUL2–RBX1 contacts. Although this dimer retains an accessible substrate‐binding site and efficiently engages the FNIP1 degron , its catalytic module is constrained, limiting productive E2–ubiquitin engagement and substrate ubiquitylation. Disruption of the CUL2–RBX1 dimer interface markedly enhances FNIP1 degron ubiquitylation both in vitro and in cells. Together, these findings identify an autoinhibited state of N8‐CRL2 FEM1B that efficiently recognizes the isolated FNIP1 degron but has limited activity towards this substrate. They support a model in which substrate‐dependent remodeling or dissociation of this dimer enables efficient ubiquitylation, revealing an additional layer of CRL2 FEM1B regulation after neddylation.