Fig. 2: OspC3 induces ADP-riboxanation of SEPT9 on multiple arginine residues.

a Coomassie blue-stained purified 3×FLAG-SEPT9 from 293T cells co-transfected with GFP-OspC3-WT or GFP-OspC3-EH/AA prior to MS analyses. Right: ClustalW2 multiple sequence alignment of SEPT3 subgroup members. Key salt-bridge residues at the NC-interface are marked with yellow circles. ADP-riboxanation sites are highlighted in red; asterisks indicate conserved residues. The six arginine residues labeled in black were identified after the initial four sites were mutated to lysine (4RK). b Extracted ion chromatograms of an unmodified peptide, an Arg561-containing peptide and its ADP-riboxanated form from immunoprecipitated SEPT9 in (a). c Tandem mass spectrum of the ADP-riboxanated Arg561-containing peptide acquired under collision-induced dissociation (CID). d, e Validation of MS-identified sites by site-directed mutagenesis. 293T cells co-transfected with GFP-OspC3 and FLAG-SEPT9 (WT or variants) were analyzed by IP and immunoblotting. 10RK refers to substitution of all ten modified arginine residues to lysine. f Prokaryotic co-expression. GST-SEPT9 was expressed in E. coli alone or with calmodulin (CaM) and OspC3 as indicated. Purified GST-SEPT9 was blotted to detect ADP-riboxanation. g In vitro reconstitution. Purified recombinant proteins were incubated (37 °C, 1 h) and analyzed by Coomassie staining and Western blotting. The effects of NAD ⁺ , CaM, and calcium ions were tested. h ADP-riboxanation during infection. HeLa cells transfected with FLAG-SEPT2, SEPT6, SEPT7, or SEPT9 were infected with S. flexneri (MOI = 50, 2 h). Anti-FLAG immunoprecipitates were analyzed by immunoblotting. i OspC-dependent modification of Arg561. HeLa cells expressing FLAG-SEPT9 (WT or R561 mutants) were infected with indicated S. flexneri strains (MOI = 50, 2 h). Modification was assessed by anti-FLAG IP and immunoblotting. UI, uninfected. j Endogenous SEPT9 modification. Analyzed as in (i), but immunoprecipitated with an anti-SEPT9 antibody. For (a, d–j), experiments were repeated three times with similar results.