Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction

Abstract

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.

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…S41 replaces basic residues observed in all other PtdIns-(3)P binding PX domains, while the tryptophan is atypical of the aromatic residues observed at the floor of this binding pocket (invariably tyrosine or phenylalanine). Interestingly, we observed binding of PA to Noxo1β and γ, as was reported previously with p47 phox ; this was attributed to the existence of a second anionic lipid binding pocket in p47 phox [36,45]. PA binding to Noxo1 is likely due to the remarkable structural similarities of Noxo1 within regions identified within this second site of p47 phox .…”

“…Thus, both the PX domain-membrane interaction and SH3 domain-p22 phox interaction contribute to the interaction of Noxo1 with the membrane and its assembly with the active oxidase complex, although Noxo1 serves no apparent role in localization of the Nox1-p22 phox heterodimer. A similar dual interaction with the membrane is well described in the case of p47 phox , although both interactions are prevented in the absence of cell stimulation [34,36,42]. Nuclear localization of Nox1 and of p22 phox has been reported in transformed human gingival keratinocytes [43] and in human umbilical vein endothelial cells (HUVEC) [44], respectively.…”

“…Critical phosphoinositide binding residues identified in p47 phox [36], which are thought to favor binding of PtdIns-(3,4)P 2, are not conserved in any Noxo1 isoform. For example, p47 phox residues R43, F44, T45, and R90 coincide with Noxo1β residues S41, W42, D43, and R91 (Fig.…”

“…It is likely that this peptide targets Noxo1γ to the nucleus. In p47 phox the PX domain proline-rich motif was shown to interact with the second SH3 domain within p47 phox , thereby masking the phosphatidylinositol-binding pocket and preventing this membrane interaction in the absence of cell stimulation and p47 phox phosphorylation [34,36]. Thus, the absence of a suitable SH3-binding motif in the PX-domains of all Noxo1 isoforms, in addition to the absence of sequence homologous to the phosphorylated auto-inhibitory of p47 phox , could explain why these proteins are membrane-bound even without cell stimulation.…”

“…This comparison reveals that lysine 50 in Noxo1β and γ is surrounded by highly conserved sequences in all other PX domains, which have been shown to form α-helices in the three-dimensional structures of p47 phox , p40 phox , Vam7p, and phosphoplipase D1 (PLD1) [Fig. 1C, [33][34][35][36][37]]. Thus, the absence of lysine 50 would likely have drastic effects on the overall conformation of the PX domains of the Noxo1 α and δ forms.…”