The Domain Organization of p67phox, a Protein Required for Activation of the Superoxide-Producing NADPH Oxidase in Phagocytes

Abstract

The phagocyte NADPH oxidase, crucial for innate immunity, is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. In activation of the oxidase, the multidomain protein p67^phoxplays a central role: it translocates to the membrane as a ternary complex with p47^phoxand p40^phox, and interacts with the small GTPase Rac to assemble with the membrane-integrated catalytic protein gp91^phox, leading to superoxide production. Here we show, using small-angle X-ray scattering (SAXS) analysis, that p67^phoxadopts an elongated conformation when it exists not only as a monomer but also as the heterotrimer. Although p67^phoxharbors an N-terminal TPR domain for binding to Rac and a p40^phox-interacting PB1 domain, followed by an SH3 domain that associates with p47^phox, the present model suggests that no or few apparent associations occur between the domains. The positions of the protein-interaction domains in p67^phoxcontribute to activation of the phagocyte NADPH oxidase: the first SH3 domain that is located between the TPR and PB1 domains positively regulates oxidase activation only when it is present at the correct position; the PB1 domain placed at this SH3 domain position inhibits the oxidase by interacting with p40^phox.