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

Abstract

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 on which we 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.

Arabidopsisinositol polyphosphate kinases regulate COP9 signalosome deneddylase functions in phosphate-homeostasis

“…In complemented lines, wild-type ubiquitinylation levels are noted. Further, in-lysate deneddylation assay to determine deneddylase defects 24 reveal decreased efficiencies of CUL1-deneddylation by endogenous CSN5 simultaneous with increased CUL1 turnover rates in ipk1-1 or itpk1-2 extracts (Fig. 2f).…”

“…Depletion of IP5K or IP6K1 reduces CRL-CSN associations in mammalian systems 24,25 . To examine whether in ipk1-1 or itpk1-2 lower CUL1 pools co-fractionate with the CSN holo-complex, extracts from these plants and Col-0 were fractionated as earlier and compared for elution profiles of CSN subunits and CUL1.…”

“…In-lysate deneddylayion assay was according to Lin et al (2020) 24 . Briefly, 14-day-old seedlings were quickly homogenized in deneddylayion buffer (10mM Tris-HCl pH7.5, 150mM NaCl and 0.5% NP-40) in absence of protease inhibitors.…”

“…Apparent contradiction between genetic requirements of the CSN-protection versus its deneddylation activity on CRLs forms the basis of the ‘CSN paradox’ 23 . In mammals, locale-specific coordinated activities of IP5K with IP6K1, functional homologs of Arabidopsis IPK1 and ITPK1, respectively provide InsP 6 to strengthen or InsP 7 to loosen CRL-CSN associations 24–26 . Interaction changes affect cellular ratio of neddylated CUL: unneddylated CUL (CUL Nedd8 :CUL) 26 .…”

Regulation of Cullin-RING E3 ligase dynamics by Inositol hexakisphosphate

“…Before explaining the results from Lin et al (5), a bit of background is necessary. Most of the current therapeutic molecular glues that target disease-causing proteins for degradation utilize the largest family of ubiquitin ligases in humans, the Cullin-RING (Really Interesting New Gene) ligases (CRLs) (6,7).…”