Cytosolic phospholipase A₂α is targeted to the p47 ^phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A₂α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67^phox or p47^phox, that cPLA ₂ specifically binds to p47^phox and that p47 ^phox is sufficient to anchor cPLA₂ to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA₂ and p47 ^phox demonstrated that the cPLA₂-C2 domain and the p47^phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA₂-C2 domain mutations were designed to explore its interaction p47^phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47^phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47^phox-PX domain caused a significant inhibition of the affinity binding to cPLA₂. Thus, Ile⁶⁷ of the cPLA₂-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47^phox-PX domain.