Extended Data Fig. 7: NEDD8 activates CRLs for millisecond poly-ubiquitin chain formation by releasing RBX1’s RING domain from cullin.

a, Graphs showing the fraction of ubiquitin-primed peptide substrates remaining with unneddylated CRL (w/o NEDD8, gray), neddylated CRL ( + NEDD8, yellow), unneddylated CRL with deletion of the cullin WHB domain (ΔWHB, light green), and CRL2s containing D660K E664K D675K CUL2 (3 K, dark green). CRL1^βTRCP2 (top-left), CRL1^FBXW7 (top-right), CRL2^FEM1C (bottom-left) and CRL2^VHL (bottom-right) E3s were examined. UB, ubiquitin. b, Graphs showing the fraction of ubiquitin-primed peptide substrates converted to ubiquitylated product as a function of UBE2R2 levels in the presence of unneddylated CRL (w/o NEDD8, gray), neddylated CRL ( + NEDD8, yellow), and unneddylated CRL with deletion of the CUL1 WHB domain (ΔWHB, light green). CRL1^βTRCP2 (left) and CRL1^FBXW7 (right) E3s were examined. c, Bar graphs showing the fraction of ubiquitin-primed peptide substrates converted to product in the presence of the E2 UBE2D3 and the indicated CRL complexes. CRL1^βTRCP2 (top) and CRL2^FEM1C (bottom) E3s were examined. d, Same as c, except with the E3s ARIH1 and CRL2^FEM1C. e, Cryo-EM map of the neddylated CRL2^FEM1C complex in the absence of UBE2R2 selected amongst multiple reconstructions. Ribbon diagrams of models corresponding to Elongin B/C-FEM1C (this study) and CUL2 (PDB code 5N4W) were fit into the density. Note the presence of additional electron density potentially corresponding to the RBX1 RING domain. f, Same as in e, except showing a cryo-EM map of the unneddylated CRL2^FEM1C complex. g, Same as in f except with ribbon diagrams of CUL2-RBX1 (top; PDB code 5N4W) or CUL1-RBX1 (bottom; PDB code 1LDJ) fit into the density. h, Superposition of CUL2-RBX1 (green and blue, respectively; PDB code 5N4W) onto the structure of a neddylated CRL1 bound to UBE2D (PDB code 6TTU, yellow). Severe steric clashing may be observed between NEDD8 and CUL2. The structural alignment was performed on the WHB domains. i, Ribbon diagram showing CUL1’s WHB domain (green) in complex with conjugated NEDD8 (yellow; PDB code 6TTU). The position of NEDD8’s Gln40 residue is shown. j, Graph corresponding to ubiquitylation reactions with ubiquitin-primed Sil1 peptide substrate and either unneddylated CRL2^FEM1C (w/o NEDD8, gray), neddylated CRL2^FEM1C (yellow), or neddylated CRL2^FEM1C harboring Q40E NEDD8 (red). Graphs are representative of triplicate (a,b and j) or duplicate (c and d) technical replicates. w/o, without.

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