Fig. 3: The SET interface is critical for 2A binding.
a Surface view of the binding interface and the selected mutants for functional validation. Residues making up the interaction interface on SETD3 and 2A are labelled in red. Residues on SETD3 mutated for further analysis are indicated in yellow. See also Supplementary Table 2. b Characteristics of the residues selected for mutations. aContact defined as <4.1 Å. See also Supplementary Table 2. c, d In vitro biolayer interferometry binding assay results for purified SETD3 variants to 2A protein. See also Supplementary Fig. 5. c Binding curves for varying input concentrations (different colours indicated in legend to individual plots) of selected SETD3 variants. Equilibrium constants KD as calculated by the integrated software of the Octet RED384 instrument are shown. d Summary table of in vitro binding assay. Rate constants and equilibrium constants were determined using the integrated software of the Octet RED384 instrument. n.d. not determined; #Binding curves showed reduced binding compared to SET257+266 mutant. Exact values could not be calculated for this low-affinity binding. e Co-AP-WB analysis of SETD3 variants binding to CV-B3 2A. Input and eluates after FLAG-affinity purification (AP) are shown. Vertical black lines indicate spliced images across Western Blot membranes; molecular weight marks are indicated on both sides for these. f Quantitative MS analysis of SETD3 variants binding to co-transfected CV-B3 2A. Protein intensities for SETD3 and 2A, as determined by label-free quantitative MS, are shown for four biologically independent experiments. Bars and error bars represent mean and standard deviation, respectively. Source data are provided as a Source Data file. Statistical models implemented in MSstats (version 4.2.0) were used to calculate P-values, which were adjusted with Benjamini Hochberg. ****P ≤ 0.0001 (P = 0.00000931, P = 0.00000041 and P = 0.0000000417, respectively).
