Binding of the PX domain of p47(phox) 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 specificities: 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 phospholipid-binding pockets radically increases membrane affinity. 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-specific 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 affinity than the isolated PX domain.

Fig. 1. The structure of the p47^phox PX domain. The ribbon representation is coloured from red at the N-terminus to blue at the C-terminus. Selected residues are shown in stick representation. Residues in the phosphoinositide pocket are shown in red and those lining the second anion-binding pocket are coloured magenta. The sulfates bound in the two pockets are shown in CPK representation.

Fig. 2. A comparison of the phosphoinositide-binding pockets of the p47^phox and p40^phox PX domains. The C_α atoms of the two domains were superimposed. The p47^phox-PX is shown as a grey ribbon, while for p40^phox the main chain ribbon (cyan) is shown only in the region of the phosphoinositide-binding pocket where the structures differ greatly. The side chains of residues that are critical for phosphoinositide binding are shown, with residue numbers for p40^phox in parentheses. The side chain of Pro78 that protrudes into the binding pocket in p47^phox is also illustrated. The bound PtdIns3P from the p40^phox-PX structure is also shown (coloured yellow, red and cyan for carbon, oxygen and phosphorus, respectively). The sulfate in the phosphoinositide-binding pocket of p47^phox-PX (grey bonds and magenta atoms) coincides with the location of the 3-phosphate of PtdIns3P. Arg42 that is associated with a CGD mutation points away from the binding site and has a role in stabilizing p47^phox (Heyworth and Cross, 2002).

Fig. 3. Interactions with the sulfates bound in the phosphoinositide-binding and second anion-binding pockets of p47^phox-PX. Dashed lines represent hydrogen bonding interactions with the protein. Ordered waters associated with the sulfates are shown as red spheres.

Fig. 4. Solid representations of the p40^phox and p47^phox PX domains. (A) View of the PtdIns3P (magenta) bound to the p40^phox-PX. The principal ligands of the phosphoinositide, Arg58 and Arg105, are highlighted in blue, and Tyr59 at the bottom of the pocket is shown in yellow. (B) View of the membrane-binding surface of the p47^phox-PX showing both phospholipid-binding sites with basic residues highlighted in blue. The sulfate in the phosphoinositide-binding site is shown in stick representation (coloured cyan). PtdIns3P from the superimposed structure of the p40^phox-PX is illustrated (stick representation). The 3-phosphate of the PtdIns3P coincides with the bound sulfate in the p47^phox phosphoinositide pocket. The Pro78 that sterically clashes with the PtdIns3P is highlighted in brown. The sulfate in the second anion-binding site is shown in CPK representation (coloured red and brown). (C) A model of a PtdIns(3,4)P₂ bound in the p47^phox-PX phosphoinositide-binding pocket. To avoid steric clashes, the inositide ring has been rotated ∼90° while maintaining the 3- and 4-phosphates in contact with the side chains of Arg43 and Arg90, respectively. The 4-phosphate (yellow) is extending into the crevice between β2 and α2. This crevice is filled by residues 97–100 in p40^phox [as seen in (A)].

Fig. 5. Sequence alignment of several PX domains. The secondary structure elements and residue numbers of p47^phox are shown above its sequence. The p47^phox residues shown by mutagenesis to be involved in membrane binding and the p40^phox residues that interact with the bound PtdIns3P are underlined. The basic residues in the second anion-binding pocket of p47^phox are marked with asterisks. The regions containing the strongly conserved (R/K)-(R/K)-(Y/F) and PXXP motifs are boxed with thin lines. The portions of p40^phox, p47^phox and Vam7p that structurally superimpose the best are enclosed in boxes with thick lines. The sequences of PLD1 and PLD2 suggest that their PX domains, like p47^phox, may have a second anion-binding pocket.

Fig. 6. Determination of the K_d for p47^phox-PX by equilibrium binding measurements. (A) Binding isotherms for p47^phox-PX and POPC/POPE/PtdIns(3,4)P₂ (77:20:3) vesicles from equilibrium SPR measurements. A solid line represents a theoretical curve constructed from R_max (45 ± 0.1) and K_d (38 ± 0.1 nM) values determined by non-linear least-squares analysis of the isotherm using an equation, R_eq = R_max/(1 + K_d/P₀). (B) Binding isotherm for p47^phox-PX and beads coated with POPC/POPE/PtdIns(3,4)P₂ (77:20:3). Phospholipid-coated beads (1.5 µM bulk concentration) were incubated with ¹⁴C-labelled p47^phox-PX (10–400 nM) in 20 mM Tris–HCl buffer pH 7.4 containing 0.1 M KCl and 1 µM BSA. A solid line represents a theoretical curve constructed from n (19 ± 1) and K_d (30 ± 7 nM) values determined by non-linear least-squares analysis of the isotherm using Equation 1.

Fig. 7. Binding of p47^phox constructs to MLVs. (A) The proteins were incubated with MLVs containing 1 mM total lipid of POPC:POPE (50:50), POPC:POPE:POPA (47.5:47.5:5), POPC:POPE:PtdIns(3,4)P₂ (47.5:47.5:5) and POPE:POPE:POPA:PtdIns(3,4)P₂ (45:45:5:5). P and S indicate ‘pellet’ and ‘supernatant’ fractions after centrifugation. Samples were analysed by SDS–PAGE, and three representative gels for the p47^phox-PX, p47^phox full-length wild-type, and p47^phox full-length with the W263R mutation in the C-SH3 are shown. (B) The intensities of the stained ‘pellet’ bands were quantitated by densitometry, and the percentage of protein bound was determined. The values represent at least four independent measurements. (C) SDS–PAGE analysis of His₆-p47^phox constructs used in the binding studies. An estimated 0.5 µg of each protein was loaded on a 4–20% gradient SDS–polyacrylamide gel. The gel was stained with SimplyBlue SafeStain. All proteins were expressed with a His₆ affinity tag, and purified as described.

Fig. 8. Activation-dependent membrane binding of p47^phox. In the resting state, p47^phox is locked in a closed state by an intramolecular interaction between the C-SH3 and the PXXP motif of the PX domain, preventing phospholipid binding. Phosphorylation of sites in the C-terminal tail releases the lock, freeing the PX domain to bind to membranes with both PX phospholipid-binding pockets and a basic region in the C-terminal tail.