Figure 5: Re-engineering the PCGF4–RING1B ligase for high activity.
From: BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase
(a,b) N-terminal swap with PCGF5 (P4 S; Fig. 2c) and helix α3 mutations (P4 M; Fig. 4) were evaluated individually or combined (P4 SM) in the wild-type background of PCGF4. E3 activity of the RING1B complex was assessed in the auto-ubiquitination assay (a) or the E2 discharge assay (b) as visualized by western blot. ‘*’ marks a band that appears to be UbcH5c conjugated to diUb. (c). E2 discharge quantified by FP. Data are plotted as pseudo first-order rate constants (k′±s.d.) versus lysine concentration. Averages were determined from duplicate measurements for all PCGF4 variants. Second-order rate constants (slopes of the linear fits shown) are reported in Table 1.
