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(phax) with P22(phax), a subunit of the oxidase catalytic core flavocytochrome b(558). Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of P47(phax) sandwich a short PRR (proline-rich region) of p22(phax) (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(phax), as expected from the model, is required for oxidase activation. Deletion of the linker between the P47(phax) SH3 domains results not only in a defective binding to p22(phax) but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro(152), Pro(156) and Arg(158) in the p22(phax) PRR as residues indispensable for the interaction with P47(phax) Pro(152) and Pro(156) are recognized by the N-terminal SH3 domain, whereas Arg(158) contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22(phax) PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47(phax) with p22(phax) thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22(phax) (amino acids 161-164), adopting an alpha-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47(phax) SH3 domains.
