Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47^phox, p67^phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: An essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b₅₅₉ with three cytosolic components: p47^phox, p67^phox, and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b ₅₅₉ within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47^phox, the tetratricopeptide repeat and activation domains of p67^phox, and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC ₅₀) for and formed a more stable complex (longer half-life) with cytochrome b₅₅₉ compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22^phox via Trp¹⁹³ in the N-terminal SH3 domain of the p47^phox segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67^phox and Rac1 segments complementary to their physical fusion. The PX domain of the p47^phox segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.