Membrane Binding Mechanisms of the PX Domains of NADPH Oxidase p40 and p47

Abstract

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).

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 .…”

Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase Cδ

“…In accordance with this, W180G and L182G had 8-and 6-fold lower affinity for POPC/POPS/ DiC 18 (59:40:1) vesicles than wild type, respectively, whereas W252G and L254G had wild type-like vesicle affinity (see Table I). Both W180G and L182G exhibited an increase in k d , which is consistent with the putative involvement of the mutated hydrophobic residues in membrane penetration (45,56,57). Monolayer measurements further supported this notion.…”

“…The higher affinity for PS-containing vesicles is attributed to a 1.7-fold increase in k a and a 3.2-fold decrease in k d . It has been shown that membrane dissociation is slowed by short-range specific interactions or hydrophobic interactions achieved by the membrane penetration of protein (45,56,57). Thus it appears that the serine head group of PS interacts specifically with PKC␦ or is able to facilitate membrane penetration of protein (or both).…”

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

“…The FYVE domain interaction with acidic membrane lipids other than PtdIns(3)P not only enhances the membrane affinity but also stabilizes a more interfacially active orientation where the PtdIns(3)P molecule is sandwiched between the domain and membrane. Likewise, membrane binding of p40 phox and p47 phox PX domains (16,45), ENTH domains of Epsin1 and the analogous ANTH domain of AP180 (46), and the PH domain (47,48) of phospholipases C-␦, C-␥, and C-␤ (49) is enhanced by the nonspecific electrostatic contacts with acidic lipids in membranes.…”