Fig. 5: The charge at Q588 dictates ubiquitin binding and UBE3A activity.
a Heat plot showing normalized BAR values of mutations at position 588 in UBE3A. White shading represents WT UBE3A activity levels, blue shading indicates loss-of-function, and red shading indicates gain-of-function. Scale bar shows the percent change relative to WT UBE3A. N = 3 independent experiments for Q588S, Q588T, Q588N, Q588K, Q588P, Q588M, Q588G, Q588A, Q588F, Q588Y, Q588W, Q588L, Q588V, Q588I, Q588C; n = 8 for Q588E, n = 6 for Q588D, n = 5 for Q588R, n = 4 for Q588H, *p < 0.05, **p < 0.005, ***p < 0.0005, One-sample t-test (two-tailed) with Benjamini–Hochberg multiple comparison correction (FDR = 0.05). b, c Fluorescence polarimetry to obtain dissociation constants (KD) for ubiquitin binding to UBE3A. d, e Schematic (d) and representative images (e) from rolling magnetic probe assays with beads conjugated to ubiquitin rolling on coverslips coated with WT, Q588E, and Q588R HECT domains from UBE3A. Dashed circle represents bead position at t = 0 and arrowhead represents bead position t = 5 s after a magnetic field was applied. Scale bar = 25 μm. f Plot showing mean ± SE of rolling parameters (ζ) of different conditions. H2O and phosphate buffered saline (PBS) were used as negative controls and biotinylated beads rolling on streptavidin-coated coverslips were used as a positive control. Note the ζ of Q588R is near negative control values. WT, n = 58; Q588E, n = 71; Q588R, n = 42, ***p < 0.0001, One-way ANOVA with Tukey’s multiple comparisons test.
