Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL–CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP₆) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP₆ in complex with CSN subunit 2 (CSN2), based onwhichwe identified the IP₆-corresponding electron density in the cryoelectron microscopy map of a CRL4A–CSN complex. IP₆ binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL–CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP₆ binding-deficient Csn2K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP₆ small molecule, whose metabolism may be coupled to CRL–CSN complex dynamics.