Figure 1 |. Role of NEDD8 and strategy to visualize dynamic ubiquitin transfer from E2 UBE2D to substrate by neddylated CRL1^β-TRCP.

a, Effect of CUL1 neddylation on CRL1^β-TRCP-catalyzed UB transfer from E2 UBE2D to a radiolabeled β-catenin-derived peptide substrate. Plots show substrate remaining during pre-steady-state rapid quench-flow ubiquitylation reactions with saturating UBE2D3 and either unneddylated or neddylated CRL1^β-TRCP. The symbols show the data from independent experiments (n = 2 technical replicates). b, Cartoon representing substrate priming by neddylated CRL1^β-TRCP and UBE2D~UB. Inset shows transition state during ubiquitylation. c, Chemical mimic of the ubiquitylation intermediate, where surrogates for the active site of UBE2D, the C-terminus of UB, and the UB acceptor site on the IκBα-derived substrate peptide are simultaneously linked.