Fig. 3: Cryo-EM and biochemical analysis of CUL9 variants reveal E2 binding, locations of DOC domains and cullin–RING and RBR elements essential for ubiquitylation activity.
a, The top left shows a cartoon schematic of hexameric CUL9–RBX1 assembly with color-coded protomers. The center shows the structure of CUL9–RBX1 protomers A, B and C with E2 enzyme docked and colored as in Fig. 2. The right shows a close-up of RING1-E2-ARM3 interactions, displayed in cryo-EM density. Quality of density allows fitting of E2 enzyme structure but was not sufficient to determine E2 identity. b, In vitro ubiquitylation assays testing autoubiquitylation and activity toward substrate TP53, comparing reactions with UBE2L3 and UBE2D2 as E2s, role of CUL9 RBR Rcat with catalytic C2294A substitution and of CUL9 WHB domain neddylation with K1881R substitution. Assays detect fluorescently labeled ubiquitin (*Ub) (n = 2 technically independent experiments). c, Cryo-EM map of CUL9–RBX1 variant in which CPH domain was replaced by a GSGSGSGS linker sequence (∆CPH). For reference, unassigned central density found in WT CUL–RBX1 and in this and several other variants is circled. d, Cryo-EM map of CUL9–RBX1 variant in which ARM9 domain was replaced by a GSGSGSGS linker sequence (∆ARM9). For reference, density corresponding to RBR domain in WT CUL9–RBX1 and variants is circled. e, Cryo-EM map of CUL9–RBX1 variant lacking ARIH-RBR element (∆ARIH-RBR) by truncation at residue 1978. f, Cryo-EM map of CUL9∆DOC–RBX1. g, DOC domains fitted into the unassigned central density in CUL9–RBX1 hexamer map at low threshold. h, In vitro ubiquitylation assays testing activity of recombinant CUL9–RBX1 and CUL9–RBX1 variants. The assays were performed with either APEX2 or TP53 as substrates, and detect fluorescently labeled ubiquitin (n = 2 technically independent experiments). APEX2 was either coexpressed and copurified with CUL9–RBX1, or purified and separately added as indicated.
