Fig. 3: AriA interacts with Ocr in an ATPase-dependent manner, leading to the release of AriB from the PARIS complex.

a, Ocr-Strep pulls down AriA, but not AriB. A mutation in the ATPase active site of AriA (K39A) blocks binding to Ocr-Strep. b, Ocr releases AriB (E26A) from WT AriA (lane 3), but Ocr does not trigger AriB release from an ATP-binding-deficient variant of AriA (K39A) (lane 5). c, A 4.4-Å-resolution reconstruction of AriA that was purified using Ocr-Strep as a bait. The structure shows three radial pores symmetrically positioned around a central pore. The pores contain disordered loops with several positively charged residues. Ocr (PDB: 1S7Z) is shown as an electrostatic surface. d, Mutations in the central pore (R116A or R119A) reduce the efficiency of phage protection. e, Ocr-Strep binds WT AriA and ejects AriB (E26A), and the charge swap mutations in the central pore of AriA (R116E/R119E) limit interaction with Ocr-Strep. f, AriB-Strep (E26A) pulls down WT AriA but not AriA (R116E/R119E). g, ATP and ADP were separated using thin-layer chromatography (Extended Data Fig. 7), and the rates of ATP hydrolysis for PARIS, PARIS mixed with tenfold excess Ocr and PARIS with an ATPase mutation in AriA (E393A) were measured. Experiments were performed in triplicate, and error bars show ±s.e.m. Two-sided statistical analysis performed using a post hoc Dunnett’s test, ****P < 0.0001. h, Size exclusion chromatography of AriB-Strep (around 36 kDa) following Ocr-mediated release from AriA. The column was calibrated using molecular weight standards (grey lines). AriB elutes in a single peak with an estimated molecular weight of 81 kDa (Extended Data Fig. 5a). i, Glutaraldehyde (GA) cross-linking assay with activated AriB (E26A). j, Mutations predicted to block AriB dimerization (E285R and F137A) prevent PARIS-mediated defence (Extended Data Fig. 1g). Plaque assays were performed in duplicate with two independent clones of each mutant.