Extended Data Fig. 5: Structural and functional analysis of AriA-AriB complex.
From: Architecture and activation mechanism of the bacterial PARIS defence system
a, b, Two similarly oriented views of AriAEQ-AriBE90A and P. aeruginosa PA14 Wadjet complex (PDB ID 8DK3)20, illustrating that the AriB binding interface is canonically conserved among other SMC ATPases. c, ATPase activity of AriA and AriA-AriB complex. ATP hydrolysis is expressed as moles of ATP hydrolysed per minute per mole of AriA (as AriA6) or AriA-AriB (as AriA6B2). Error bars represent the average and standard deviation of three independent measurements (n = 3; open circles). d, Top: Structural overlay presenting AriAEQ and AriAEQ-AriBE90A complexes. Bottom: a close-up view depicts changes in the AriA ATPase head region upon binding with AriB. The residues involved in AriB binding are shown in stick representation for side chains. AriB is omitted for clarity. e, Cartoon representation of AriB monomer, with the toprim domain in pink and helical bundle domain in white. At the bottom; Close-up view of the AriB active site. f, Overall structure of Geobacillus stearothermophilus (Gs) M5 RNase bound to the 50 S ribosome. M5 RNase is shown in a contrast clear window, zoomed in cartoon view below (PDB ID 6TPQ)24. Bottom: Close-up view of the active site of M5 RNase. g, Cartoon structure of OLD DNase from Burkholderia pseudomallei (Bp) (PDB ID 6NK8)55. Two bound metal ions are shown as green spheres. Bottom: Close-up view of the active site. h, Overlay of active site residues from AriB, M5 RNase, and OLD DNase. The active site residues are shown in ball-and-stick representation. i, Top: A representative plaque-forming unit assay, demonstrating T7 phage plaques on the E. coli MG1655 bacterial lawn carrying either an empty vector (EV), the PARIS system (Wt; AriA-AriB as an operon), or the RNA binding mutant AriBR28E (AriA-AriBR28E). Bottom: The data represent mean and standard deviation of plaque-forming units (PFU) mL−1 of phage T7 from three independent replicates, with individual data points shown (n = 3). j, Bacterial dilution spotting (10-fold) assay performed to test the toxicity comparison in AriB and its point mutants (E90A and R28E). Data shown are representative of three replicates (n = 3). k, SDS-PAGE analysis of coexpression of AriA-AriB and AriA-AriBR28E constructs followed by Ni2+ affinity purification of AriA and associated proteins. Data are representative of three independent experiments.
