Figure S7.

Roles of CAND1 across cullin-RING ligase systems, related to Figure 7

(A) Heatmap showing percentages of FLAG-CUL1-associated versus total Fbps at steady state from WT or CAND-null cells endogenously expressing FLAG-tagged CUL1,⁹ or the CAND-null cells stably expressing WT CAND1 or the indicated β-hairpin mutants.

(B) Immunoblotting blotting for components of SCF^BTRC at steady state, which align with the proteomics results shown in Figure 7B. Chemiluminescent signal with adjusted brightness and contrast is shown.

(C) Docking of SKP1-SKP2 crystal structure⁴⁶ onto CAND1-SCF^FBXW7 rolling-2c conformation shows potential incompatibility between SKP2 and CAND1’s anti-neddylation domain in this orientation.

(D) Cryo-EM structures show addition of substrate complex (phosphorylated p27-CKSHS1-CyclinA-CDK2) alters distribution of CAND1-SCF^SKP2 conformations, including SCF^SKP2 complexes free of CAND1.

(E) Overlay of various crystal structures of CUL-substrate receptor complexes with the CAND1 engaged/SCF^SKP2 rocked structure. Structures were superimposed over CUL CR1 domains. The modeling shows clashing of CAND1’s β-hairpin with CUL2,⁵⁶ CUL3,⁶² and CUL5⁶³ substrate receptors, suggesting that their disassembly and assembly mechanisms may parallel that described herein for SCFs.

(F) Alignment of CAND1-CUL4-RBX1¹⁴ (left) and cryo-EM structure of CAND1-CUL1-RBX1 (right) on CUL4-bound DDB1,⁵⁵ superimposed over the CUL N-terminal CR1 domains, highlighting positions of CAND1’s β-hairpin and C-terminal HEAT repeat loop.