p47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different speci®cities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40 phox , while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipidbinding pockets radically increases membrane af®n-ity. Strikingly, measurements for full-length p47 phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-speci®c mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater af®nity than the isolated PX domain.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.

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