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The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1–PB1 association with p67phox. [ABSTRACT FROM AUTHOR]

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67phox and p47phox. They translocate to the membrane upon cell stimulation and activate flavocytochrome b558, the membrane-integrated catalytic core of this enzyme system. The activators p67phox and p47phox form a ternary complex together with p40phox, an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif-containing domains: p40phox associates with p67phox via binding of the p40phox PC motif to the p67phox PB1 domain, while p47phox directly interacts with p67phox but not with p40phox. Here we show that p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40phox carrying the D289A substitution in PC or a p67phox with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40phox participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67phox and p47phox via the PB1-PC interaction with p67phox. [ABSTRACT FROM AUTHOR]

PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs. We report here the NMR structure and ligand-binding site of the PB1 domain of the cell polarity establishment protein, Bem1p. In addition, we identify the topology of the PC motif-containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p. The PC motif-containing region is a structural domain offering a scaffold to the PC motif. The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain. A structural database search reveals close similarity between the Bem1p PB1 domain and the c- Raf1 Ras-binding domain. However, these domains are functionally distinct from each other. [ABSTRACT FROM AUTHOR]