Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47^phox in recognition of a polyproline type II helix and an adjacent α-helix of p22^phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47^phox with p22^phox, a subunit of the oxidase catalytic core flavocytochrome b₅₅₈. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47^phox sandwich a short PRR (proline-rich region) of p22^phox (amino acids 151–160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47^phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47^phox SH3 domains results not only in a defective binding to p22^phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro¹⁵², Pro¹⁵⁶ and Arg¹⁵⁸ in the p22^phox PRR as residues indispensable for the interaction with p47^phox. Pro¹⁵² and Pro¹⁵⁶ are recognized by the N-terminal SH3 domain, whereas Arg¹⁵⁸ contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22^phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47^phox with p22^phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22^phox (amino acids 161–164), adopting an α-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47^phox SH3 domains.