Figure 4. NEDD8 Pushes an RBX1 Lever and Directs an RBX1 Pivot to Juxtapose the Active Site and Acceptor Lys.

(A) RBX1 RING domain and W35 “pivot” positions in neddylation complex compared with CUL1-RBX1-CAND1 (Goldenberg et al., 2004; Zheng et al., 2002), with CUL1 aligned.

(B) Close-up of contacts between UBC12-linked donor NEDD8 and RBX1 linker. Zinc atoms bound to RING are shown as spheres.

(C) As in (B) except with NEDD8 in surface colored by identity with UB. RING and UBC12-binding residues are identical between NEDD8 and UB (yellow), but exposed surfaces and contacts to RBX1 pivot differ (orange).

(D) Comparison of effects of Ala mutations in place of RBX1 “lever” (I37) or other linker residues in RBX1-mediated NEDD8 transfer from UBC12 to CUL1^CTD. Graph, rate compared to WT RBX1; error, 1 SD.

(E) Same as (D) but with variants of RBX1 Trp35 pivot.

(F) Same as (E) but monitoring fluorescent UB transfer from UBCH5B.

(G) Immunoblot comparing RBX1-mediated transfer of WT NEDD8, or variants with corresponding E31Q and E32D from UB, from UBC12 to CUL1^CTD. Graph, rate compared to WT UBC12~NEDD8; error, 1 SD.

(H) RBX1-mediated transfer of fluorescent UB, or “neddylized” variants with Q31E and D32E, from UBCH5B to CUL1^CTD. Graph, rate compared to WT UBCH5~UB; error, 1 SD.

(I) Schematic of roles and orientations of RBX1 elements and interactions with UBC12Pulse-chase assays forNEDD8, oriented as in prior RBX1-CUL1 structures or RBX1-UBC12~NEDD8-CUL1^CTD-DCN1^P.

(J and K) Pulse-chase assays for NEDD8-modifed SCF^Fbw7ΔD testing roles of RBX1 W35 “pivot” (J) and I37 “lever” (K) in directing WT or “neddylized” Q31E/D32E UB from UBCH5B to either an F-box-bound substrate (Cyclin E phosphopeptide) or neddylated CUL1.

See also Figure S3 and Movie S2.