Cryo-EM reveals an autoinhibited dimeric state of the neddylated CRL2FEM1B 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 CRL2FEM1B. Structures of N8-CRL2FEM1B in the absence and presence of the FNIP1degron reveal a crown-shaped homodimer stabilized by interprotomer CUL2-RBX1 contacts. Although this dimer retains an accessible substrate-binding site and efficiently engages the FNIP1degron, its catalytic module is constrained, limiting productive E2-ubiquitin engagement and substrate ubiquitylation. Disruption of the CUL2-RBX1 dimer interface markedly enhances FNIP1degron ubiquitylation both in vitro and in cells. Together, these findings identify an autoinhibited state of N8-CRL2FEM1B that efficiently recognizes the isolated FNIP1degron 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 CRL2FEM1B regulation after neddylation.

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