Fig 4. Specialised residue pair within the E2-fold regulates RING E3 driven substrate ubiquitination.

a, Comparison of a residue network with structures of unbound (grey, PDB ID 1yh2) and FANCL^R-bound (orange, PDB ID 1yh2) Ube2T depicting the proposed allosteric conduit. The E54 functions as a gating residue based on its influence on R69, which in turn acts as an effector residue for stabilising the catalytic beta-element. b, FANCL^UR mediated in vitro xFANCD2 ubiquitination using Ube2T mutants of the gating and effector residues. Graphs represent percentage xFANCD2 monoubiquitination (n=3) with a line at mean value. Significant differences were assessed by one-way ANOVA with Dunnett’s multiple-comparison test. Adjusted P values are shown. c, Structure of Ube2T gating mutant E54R (magenta) reveals a bonding network in the allosteric conduit similar to the FANCL^R-bound Ube2T structure. d, Monoubiquitination of xFANCD2 (1 µM) under single-turnover conditions shows reactivity of the indicated E2~Ub thioester (1 µM) in the absence or presence of FANCL^UR (1 µM). Percentage of xFANCD2 monoubiquitination (mean ± range, n=3) is plotted over time. To prevent E2 auto-ubiquitination, Ube2T^{1-152, K91R} is used and indicated mutations incorporated in this background. e, Comparison of a residue network with structures of unbound Ube2B (grey, PDB ID 2yb6) and Rad6 Binding Domain (R6BD) bound Ube2B (green, PDB ID 2ybf) depicting the proposed gating and effector roles for Ube2B residues E58 and R71, respectively. f, Immunoblots of in vitro ubiquitination assays shows Ube2B with a permissive gate (E54R) is more responsive to Rad18 in PCNA monoubiquitination, while the Ube2B effector mutant (R71A) slows the reaction. Percentage of PCNA-Ub (n=3) were quantified, linear rates normalized to Rad18-Ube2B wildtype reaction and plotted with a line at mean value. Raw images in Supplementary Fig. 11.