Fig. 6: Cullin neddylation, assembly with ARIH-family E3s and effects on deneddylation.

Schematic displaying CRL structures (surface representation) in their unneddylated, (neddylated) states and as E3–E3 super-assembly representation. a, The left shows that in cells, cullins 1–3 (represented by (PDB 1LDJ) are preferentially neddylated via UBE2M. The deneddylase CSN removes NEDD8. The center shows neddylated cullins 1–3, with a flexible NEDD8-WHB unit, can form E3–E3 super-assemblies with ARIH1 (represented by PDB 7B5L, showing only NEDD8-linked CUL1–RBX1). The right shows active neddylated CUL1–RBX1-ARIH1 E3–E3 super assembly in conformation for ubiquitin transfer from UBE2L3 to ARIH1 (PDB 7B5L, substrate receptor complex not shown). The active conformation of the E3–E3 assembly is poised to inhibit CSN-mediated deneddylation. b, The left shows unneddylated CUL5 in complex with RBX2 (PDB 6V9I), which is neddylated by UBE2F, and deneddylated by CSN. The center shows that neddylated CUL5–RBX2 assembles with ARIH2 to form an E3–E3 super assembly (PDB 7ONI, ARIH2 not shown). The right shows active CUL5–RBX2-ARIH2 E3–E3 super assembly (PDB 7ONI). The E3–E3 assembly performs ubiquitylation and blocks CSN-mediated deneddylation. c, The left shows CUL7 forms a complex with RBX1 but is not neddylated as canonical CRLs (PDB 7Z8B, SKP1-FBXW8 not shown). On the right, together with neddylated CUL1–RBX1 and SKP1-FBXW8, CUL7–RBX1 forms an active CRL–CRL E3–E3 super assembly (PDB 7Z8B). d, In this study, hexameric CUL9–RBX1 is neddylated by UBE2F, and with chimeric E3–E3 ligase activity encompassed within the CUL9 polypeptide. Recombinant WT CUL9–RBX1 was not deneddylated in vitro by CSN or SENP8, possibly restricted by NEDD8 binding to the built-in ARIH-RBR domain. Even after deletion of the protective ARIH-RBR domain, deneddylation was CSN-independent.