Journal: Nature communications

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

doi: 10.1038/s41467-022-32758-3

Figure Lengend Snippet: 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 Supplemen- tary 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 com- plex. Size exclusion column elution profile after purification and complex recon- stitution 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

Article Snippet: Protein expression and purification A codon-optimised gene for CV-B3 (Nancy strain, M33854.1; GenBank) protease 2A (WT and C107A mutant) and for human SETD3 Nature Communications | (2022) 13:5282 9 were separately cloned into E. coli expression vector 2G-T. pET His6 GST TEV LIC cloning vector (2G-T) was a gift from Scott Gradia (Addgene plasmid # 29707; http://n2t.net/addgene:29707; RRID:Addgene_29707).

Techniques: Generated, Protein-Protein interactions, Sequencing, Cryo-EM Sample Prep, Binding Assay

Journal: Nature communications

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

doi: 10.1038/s41467-022-32758-3

Figure Lengend Snippet: 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 Supple- mentary Fig. 5. c Binding curves for varying input concentrations (different col- ours 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

Article Snippet: Protein expression and purification A codon-optimised gene for CV-B3 (Nancy strain, M33854.1; GenBank) protease 2A (WT and C107A mutant) and for human SETD3 Nature Communications | (2022) 13:5282 9 were separately cloned into E. coli expression vector 2G-T. pET His6 GST TEV LIC cloning vector (2G-T) was a gift from Scott Gradia (Addgene plasmid # 29707; http://n2t.net/addgene:29707; RRID:Addgene_29707).

Techniques: Binding Assay, Functional Assay, Biomarker Discovery, In Vitro, Software

Journal: Nature communications

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

doi: 10.1038/s41467-022-32758-3

Figure Lengend Snippet: 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 com- plex (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

Article Snippet: Protein expression and purification A codon-optimised gene for CV-B3 (Nancy strain, M33854.1; GenBank) protease 2A (WT and C107A mutant) and for human SETD3 Nature Communications | (2022) 13:5282 9 were separately cloned into E. coli expression vector 2G-T. pET His6 GST TEV LIC cloning vector (2G-T) was a gift from Scott Gradia (Addgene plasmid # 29707; http://n2t.net/addgene:29707; RRID:Addgene_29707).

Techniques: Binding Assay, Cryo-EM Sample Prep, Residue

Journal: Nature communications

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

doi: 10.1038/s41467-022-32758-3

Figure Lengend Snippet: 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

Article Snippet: Protein expression and purification A codon-optimised gene for CV-B3 (Nancy strain, M33854.1; GenBank) protease 2A (WT and C107A mutant) and for human SETD3 Nature Communications | (2022) 13:5282 9 were separately cloned into E. coli expression vector 2G-T. pET His6 GST TEV LIC cloning vector (2G-T) was a gift from Scott Gradia (Addgene plasmid # 29707; http://n2t.net/addgene:29707; RRID:Addgene_29707).

Techniques: Binding Assay, Infection, Luciferase, Expressing, Western Blot, Derivative Assay