Structural Basis of CSN-mediated SCF Deneddylation

Abstract Cullin-RING ligases (CRLs) are the largest family of E3 ligases, with their ubiquitination activity dynamically regulated by neddylation and deneddylation by the COP9 signalosome (CSN). CSN-mediated deneddylation not only deactivates CRLs but also enables substrate receptor exchange, fine-tuning CRL specificity. CSN has emerged as a promising drug target, yet the structural basis underlying its catalytic mechanism remains insufficiently understood. To address this, we used cryo-electron microscopy (cryo-EM) analysis of CSN-CRL1 (SCF) complexes to uncover distinct functional states, capturing key intermediates of the deneddylation cycle. The earliest state represents an initial docking step in which CSN remains autoinhibited. In contrast, the catalytic intermediate reveals the fully engaged state, with the CSN5 Ins-1 loop, RBX1 RING, and neddylated Cullin WHB domains repositioned for isopeptide bond cleavage. We further resolve four dissociation intermediates that define the stepwise release of CSN from its deneddylated product, highlighting a central role for RBX1 RING in stabilising key interactions throughout this process. Additionally, our structures reveal the previously uncharacterised position of CSNAP, which integrates into the CSN scaffold at a groove formed by CSN3 and CSN8. Together, our study provides a comprehensive mechanistic model of CSN function, linking CRL recognition, catalytic activation, and stepwise disengagement. These insights lay the groundwork for the rational design of CSN inhibitors, offering new opportunities to modulate CRL activity for therapeutic applications. Key Takeaways High-resolution cryo-EM structures capture key intermediates of the CSN-mediated SCF deneddylation cycle Structural identification of CSNAP places it within a previously uncharacterized groove at the interface of CSN3 and CSN8. Pre-catalytic and catalytic states define the conformational transitions of CSN5 Ins-1 , RBX1 RING , and N8 WHB that enable isopeptide bond cleavage. Four dissociation intermediates reveal a sequential pathway for CSN disengagement, with RBX1 RING playing a central role in stabilising key interactions.