Extended Data Fig. 9: Structural and functional analysis of CUL9-RBX1 neddylation and deneddylation.
a, b, Purified neddylation machinery (NEDD8 E1 (NAE1-UBA3), NEDD8 E2 UBE2F) and the indicated CUL9-RBX1 complexes were used for in vitro neddylation assays, detecting fluorescently-labeled NEDD8 (*NEDD8) in SDS–PAGE gels (n = 2 technically independent experiments). a, CUL9-RBX1ΔARIH-RBR contains residues 1–1978 and lacks the ARIH-RBR element, CUL9-RBX1K1881R-ΔARIH-RBR is the same construct with the neddylation site K1881R substitution. b, CUL7-CUL9-RBX1 4HB-to-WHB chimera has the CUL7 sequence with the region spanning from the 4HB domain to the WHB domain swapped with the CUL9 sequence and CUL9-RBX1-CUL7 4HB-to-WHB chimera is the reciprocal swap of these domains. c, Treatment of parental U2OS cells, and CUL9 knock-out cells exogensously expressing CUL9 with CSN inhibitor (CSNi) or DMSO as control. Immunoblots detect NEDD8 (N8) linked to CUL9 or canonical cullins (CUL1-5), or total CUL9 or Vinculin as loading control (n = 2 technically independent experiments). d, e, Overlay of CSN-CUL2-RBX1 complex (PDB: 6R7N) and CUL9-RBX1 (this study) aligned on CR3 and 4HB of neddylated CUL9-RBX1 protomer. d, CSN modeled on neddylated protomer of mixed CUL9-RBX1 dimer is shown. e, CSN modeled only on neddylated protomer is shown, with closeup in inset on the right.
