Structure-function analysis of enterovirus protease 2A in complex with its essential host factor SETD3

Figures

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  Fig. 1. CV-B3 2A and SETD3 form a stable complex. a High-confidence SETD3 interacting host proteins in the presence and absence of CV-B3 2A with a Bayesian False Discovery Rate (BFDR) < 0.05 generated using AP-MS. See also Supplementary Dataset 1. b Schematic of sequential FLAG-Strep AP (double-AP) experimental flow. c Dot plot representation of proteins identified in double-AP experiment. Sequence coverage of detected proteins versus SAINTexpress BFDR is shown. Proteins passing the significance threshold of BFDR < 0.05 are highlighted in red. See also Supplementary Dataset 2. d Purification of reconstituted SETD3-2A complex. Size exclusion column elution profile after purification and complex reconstitution is shown. Aggregated material (peak 1), the complex of 2A-protease with SETD3 (peak 2), and monomeric 2A (peak 3). Purified complex from peak 2 was used for structure determination by cryo-EM. See also Supplementary Fig. 1. e Binding of purified SETD3 to 2A protein as measured by biolayer interferometry. See also Supplementary Figs. 1–5. Binding curves for varying input concentrations (different colours) of SETD3. Equilibrium constant KD as calculated by the integrated software of…

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  Fig. 2. Structure of CV-B3 2A and SETD3 in complex. a Cryo-EM map and ribbon representation of SETD3-2A complex. The SET domain of SETD3 is shown in purple, the linker in grey, the RSB domain in cyan, and CV-B3 2A protease in orange. See also Supplementary Tables 1 and 2, Supplementary Figs. 2–4. b, c Expanded views of SET interface and RSB interface interactions, with important residues highlighted. See also Supplementary Tables 1 and 2, Supplementary Figs. 2–4. Indicated contact residues of SETD3 and 2A are shown as stick representation. SETD3 residues selected for mutational studies (see Fig. 3 and onwards) are labelled in bold font.

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  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…

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  Fig. 4. 2A binding partially overlaps with the substrate binding site at the SET interface. a Superposition of SETD3-2A cryo-EM structure and SETD3-actin complex (PDB ID 6mbk) with SETD3 from actin complex not shown. SETD3-2A ribbon model colours as in Fig. 2, β-actin bound to SETD3 is coloured in green. S-adenosyl-l-homocysteine (SAH) in yellow, actin substrate residue H73, and SETD3 Y313 are shown as stick models located in the active site of SETD3. Inset shows details of the intermolecular β-sheet structure observed between CV-B3 2A protease and SETD3 and between β-actin and SETD3, with important contact residues and hydrogen bonds indicated in the respective colours. b Western blot analysis of actin methylation activity of WT, SETD3KO, and SETD3KO H1-Hela cells stably complemented with the indicated SETD3 variants. The expression of SETD3 mutants is shown by Western blot. c Impact of actin peptide on SETD3-2A binding in biolayer interferometry assay. Maximum SETD3 binding (310 nM) is plotted against peptide concentration. n = 7 independent binding events at different peptide concentrations were measured over four independent experiments. Data are presented as mean values ± SD over…

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  Fig. 5. 2A binding at the SET interface is required for viral infection. See also Supplementary Fig. 7. a Schematic of synchronised luciferase-expressing CV-B3 (MOI 1) infection. Graphic created with BioRender.com. b Western blot analysis of H1-Hela WT, SETD3KO or SETD3KO cells complemented with SETD3 variants under a minimal promoter are shown. c. Infection of WT, SETD3KO or SETD3KO cells complemented with SETD3 structure-derived mutants. n = 3 biologically independent samples were measured for one independent experiment. Data are presented as mean and standard deviation. Source data are provided as a Source Data file. Statistics were performed on 8 h timepoints using GraphPad Prism 9 (Version 9.3.1). P-values were determined by two-way ANOVA (Holm–Sidak corrected) on log-transformed data. RLU relative light units, n.s. not significant, ****P ≤ 0.0001.