Membrane Binding Mechanisms of the PX Domains of NADPH Oxidase p40phox and p47phox*

Robert V. StahelinDagger , Aura BurianDagger , Karol S. Bruzik§, Diana Murray, and Wonhwa ChoDagger ||

From the Departments of Dagger  Chemistry and § Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60607 and the  Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

Received for publication, December 10, 2002, and in revised form, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40phox and p47phox, contain PX domains. The PX domain of p40phox specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47phox 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 p40phox PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47phox 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 p40phox and p47phox proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47phox, whereas such an interaction is absent in p40phox.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositides (PI)1 play pivotal roles in cell signaling and membrane trafficking by serving as site-specific membrane signals to modulate the intracellular localization and/or biological properties of effector proteins. Since the discovery of the pleckstrin homology (PH) domain as an effector for PIs, many PI-binding domains, including FYVE (Fablp, YOTB, Vac1p, and EEA1), ENTH (epsin N-terminal homology), FERM (four-point one, ezrin, radixin, moesin), and PX (Phox) domains, with different PI specificities and affinities have been identified. The PX domain was initially identified as a conserved structural module of about 130 amino acids in the p40phox and p47phox subunits of the NADPH oxidase complex (1). The PX domain has been since found in over 100 eukaryotic proteins (2-6). As is the case with the PH domain, PX domains show broad PI specificity. Whereas many PX domains, including those from p40phox (7, 8), Vam7p (9), and sorting nexin 3 (10), have been found to be specific for phosphatidylinositol 3-phosphate (PtdIns(3)P), PX domains from p47phox (11, 12), yeast Bem1p (13), and Class II PI 3-kinase (14) preferentially bind to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate, respectively.

NADPH oxidase, which is activated upon phagocytosis of foreign particles, plays an instrumental role in the host defense mechanism of neutrophils and macrophages. NADPH oxidase is a multisubunit protein complex consisting of cytosolic components p40phox, p47phox, p67phox, and Rac as well as a membrane-bound heterodimer of p22phox and gp91phox. When invasive microorganisms are sequestered, the cytosolic subunits translocate to the membrane surface and fuse with the membrane-bound subunits, creating the activated complex (15). Activated NADPH oxidase then produces superoxide, which in turn generates microbicidal reactive oxygen species in phagocytic vesicles. Chronic granulomatous disease, which confers severe susceptibility to infection, is due to naturally occurring mutations in the NADPH oxidase complex, including some in the p47phox subunit. p47phox is a prerequisite for assembly of the entire complex (16, 17), and the p40phox subunit is required for subunit translocation and proper functioning of the active complex (18). Both p40phox and p47phox contain PX domains. The recently determined x-ray crystal structures of these PX domains (7, 12) have revealed the basis of the stereospecific recognition of their cognate PIs (i.e. PtdIns(3)P for p40phox-PX and PtdIns(3,4)P2 for p47phox-PX). However, the PI-dependent membrane-binding mechanisms of these PX domains as well as their roles in NADPH oxidase activation are relatively poorly understood. Evidence indicates that these PX domains have distinct membrane binding properties. For instance, the p47phox-PX has a unique secondary binding site that nonspecifically binds anionic phospholipids, including phosphatidic acid (PtdOH) and phosphatidylserine (PtdSer). Furthermore, the p40phox-PX was shown to be localized at PtdIns(3)P-rich endosomes when expressed ectopically in mammalian cells (7, 19), whereas the isolated p47phox-PX exhibited cytoplasmic distribution under similar conditions (19).

This study was undertaken to determine the origin of differential membrane targeting mechanisms of p40phox-PX and p47phox-PX, with an emphasis on elucidating the roles of PtdIns(3)P and PtdIns(3,4)P2 in their membrane binding. Results from in vitro membrane binding measurements by monolayer and surface plasmon resonance (SPR) analyses, electrostatic potential computation, and subcellular localization measurements using enhanced green fluorescent protein (EGFP)-tagged domains provide new insight into how differently PtdIns(3)P and PtdIns(3,4)P2 mediate the membrane targeting of p40phox-PX and p47phox-PX, respectively, which in turn provide an important clue to the understanding of the membrane recruitment mechanisms of p40phox and p47phox subunits of NADPH oxidase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE) were from Avanti Polar Lipids (Alabaster, AL). 1,2-Dipalmitoyl derivatives of PtdIns(3)P, phosphatidylinositol-5-phosphate, PtdIns(3,4)P2, and phosphatidylinositol-4,5-bisphosphate were synthesized as described elsewhere (20). These derivatives were selected over naturally occurring PIs containing cis-unsaturated sn-2 acyl groups due to difficulties involved in the chemical synthesis of the latter. Since the concentration of PIs used in this study was limited to 3 mol %, the effect of their sn-2 acyl chains on the membrane structure would not be significant. Phospholipid concentrations were determined by phosphate analysis. The Liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Canada). Fatty acid-free bovine serum albumin was from Bayer, Inc. (Kankakee, IL). Restriction endonucleases and other enzymes for molecular biology were from New England Biolabs (Beverly, MA). (3-[3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate and octyl glucoside were from Sigma and Fisher, respectively. The Pioneer L1 sensor chip was from Biacore AB (Piscataway, NJ).

Mutagenesis and Protein Expression-- Mutations of p40phox-PX were performed by the overlap extension PCR method (21). Each construct was subcloned into the pET21a vector containing a C-terminal His6 tag and transformed into DH5alpha cells for plasmid isolation. After verifying the DNA sequence of each construct, the plasmid was transformed into BL21(DE3) cells for protein expression. One liter of 2× YT medium containing 100 µg/ml ampicillin was inoculated with BL21(DE3) cells harboring each construct and grown at 37 °C until absorbance at 600 nm reached 1.0. At this time, 1 mM of isopropyl-1-thio-beta -D-galactopyranoside was added, and cells were then incubated at 25 °C for 5 h. Cells were harvested for 10 min at 4000 × g, and the resulting pellet was resuspended in 10 ml of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl, 50 µM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 10 mM imidazole. The solution was then sonicated for 8 min using a 30-s sonication followed by 30-s cooling on ice. This was followed by centrifugation at 48,000 × g to separate the soluble and insoluble fractions. The supernatant was filtered into a 50-ml tube, and 1 ml of nickel-nitrilotriacetic acid solution (Qiagen, Valencia, CA) was added. The mixture was incubated on ice with gentle stirring (80 rpm) for 1 h. After this time, the mixture was poured onto a column, which was washed with 20 ml of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl and 15 mM imidazole. Subsequently, the protein was eluted from the column in six fractions using 0.5 ml of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl and 300 mM imidazole. Purity was checked on an 18% polyacrylamide gel, and samples were pooled and concentrated to 1 ml for gel filtration chromatography. The 1-ml sample was loaded onto a Superdex 200 column (Amersham Biosciences) and eluted with 20 mM Tris, pH 7.5, with 0.1 M NaCl. Fractions corresponding to the p40phox-PX peak, as checked electrophoretically, were pooled and concentrated to 1 ml. Protein concentration was then determined using the BCA method (Pierce). The full-length human p40phox (residues 2-339) and W207R mutation were expressed and purified as described (7). Mutants of p47phox-PX and full-length p47phox were constructed, expressed, and purified as previously described (12).

EGFP Constructs-- EGFP in pEGFP vector (Clontech) was modified by PCR to remove the first methionine and to add two amino-terminal glycines and an EcoRI site. The modified EGFP gene was inserted into a modified pIND vector (Invitrogen) between the EcoRI and XhoI sites to yield a plasmid, pIND/EGFP. p40phox-PX was cloned by PCR to remove the stop codon and add two C-terminal glycines and an EcoRI site. The PCR product was digested with NotI and EcoRI and ligated into pIND/EGFP to generate a C-terminal EGFP-fused p40phox-PX with spacer sequence GGNSGG. EGFP-fused constructs of p40phox-PX mutants, p47phox-PX, and p47phox-PX mutants were generated by the same method.

Monolayer Measurements-- The penetration of p40phox-PX and p47phox-PX into the phospholipid monolayer was measured by monitoring the change in surface pressure (pi ) at constant surface area using a 10-ml circular Teflon trough and Wilhelmy plate connected to a Cahn microbalance as previously described (22). All our monolayer measurements were performed at 23 °C, since at higher temperature water evaporation and protein instability caused significant experimental problems. A lipid monolayer containing various combinations of phospholipids was spread onto the subphase composed of 10 mM HEPES, pH 7.4, containing 0.1 M NaCl until the desired initial surface pressure (pi 0) was reached. After the signal stabilized (~5 min), 30 µg of proteins were injected, and the increase in surface pressure (Delta pi ) was monitored for 45 min while stirring the subphase at 60 rpm. Typically, the Delta pi value reached a maximum after 20 min. It has been shown empirically that Delta pi caused by a protein is mainly due to the penetration of the protein into the lipid monolayer. For example, in the case of the C2 domain of cytosolic phospholipase A2, excellent agreement was found between large Delta pi caused by several residues in the calcium binding loops (22) and their actual membrane penetration measured by fluorescence (23) and electron spin resonance studies (24, 25). The maximal Delta pi value depended on the protein concentration and reached a saturation value (e.g. [p40phox-PX] >=  2.0 µg/ml); therefore, protein concentration in the subphase was maintained above such values to ensure that the observed Delta pi represented a maximum value. The resulting Delta pi was plotted versus pi 0, from which the critical surface pressure (pi c) was determined as the x-intercept (26).

Kinetic and Equilibrium SPR Experiments-- All SPR measurements were performed at 23 °C. A detailed protocol for coating the L1 sensor chip has been described elsewhere (27, 28). Briefly, after washing the sensor chip surface, 90 µl of vesicles containing various phospholipids (see Table I) were injected at 5 µl/min to give a response of 4000 resonance units. Similarly, a control surface was coated with vesicles, typically without the PI of interest, to give the same resonance unit response as the active binding surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for the PX domains. Each lipid layer was stabilized by injecting 10 µl of 50 mM NaOH three times at 100 µl/min. Typically, no decrease in lipid signal was seen after the first injection. Kinetic SPR measurements were done at the flow rate of 60 µl/min. 90 µl of protein in 10 mM HEPES, pH 7.4, containing 0.1 M KCl was injected to give an association time of 90 s, whereas the dissociation was monitored for 400 s or more. The lipid surface was regenerated using 10 µl of 50 mM NaOH. After sensorgrams were obtained for five different concentrations of each protein within a 10-fold range of Kd, each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from it. The association and dissociation phases of all sensorgrams were globally fit to a 1:1 Langmuir binding model: protein + (protein binding site on vesicle) left-right-arrow (complex) using BIAevalutation 3.0 software (Biacore) as described previously (28-30). The dissociation constant (Kd) was then calculated from the equation, Kd = kd/ka. Mass transport (31, 32) was not a limiting factor in our experiments, since change in flow rate (from 2 to 60 µl/min) did not affect kinetics of association and dissociation. After curve fitting, residual plots and chi 2 values were checked to verify the validity of the binding model. Each data set was repeated three times to calculate a S.D. value. Also, Kd values were separately determined from equilibrium SPR measurements. For these measurements, the flow rate was reduced to 2 µl/min to allow sufficient time for the association phase, which in turn allows resonance unit values to reach saturating response values (Req). Req values were then plotted versus protein concentrations (C), and the Kd value was determined by a nonlinear least-squares analysis of the binding isotherm using an equation: Req = Rmax/(1 + Kd/C), where Rmax is a maximal Req value.

Electrostatic Potential Computation-- The electrostatic properties of the PX domain with and without PIs were calculated with a modified version of the program DelPhi and visualized in the program GRASP (33), as described previously (34). In the panels of Fig. 6, the electrostatic potentials are represented by two-dimensional equipotential contours in 0.1 M KCl: magenta, -6 mV; dark pink, -12 mV; light pink, -25 mV; dark blue, +25 mV; light blue, +12 mV. The electrostatic calculations performed used partial charges taken from the CHARMM27 force field (35) and spatial coordinates taken from the structures of p40phox-PX (1H6H) (7) and p47phox-PX (1O7K) (12). PtdIns(3,4)P2 and PtdSer were docked onto the p47phox-PX structure using the bound sulfate ions as guides in placing the lipid phosphate groups. Steric clashes were fixed manually.

Cell Culture-- A stable HEK293 cell line expressing the ecdysone receptor (Invitrogen) was used for all experiments. Briefly, cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 and 98% humidity until 90% confluent. Cells were passaged into eight wells of a Lab-TechTM chambered cover glass for later transfection and visualization. Only cells between the 5th and 20th passages were used. For transfection, 80-90% confluent cells in Lab-TechTM chambered cover glass wells were exposed to 150 µl of unsupplemented Dulbecco's modified Eagle's medium containing 0.5 µg of endotoxin-free DNA and 1 µl of LipofectAMINETM reagent (Invitrogen) for 7-8 h at 37 °C. After exposure, the transfection medium was removed, the cells were washed once with fetal bovine serum-supplemented Dulbecco's modified Eagle's medium and overlaid with fetal bovine serum-supplemented Dulbecco's modified Eagle's medium containing ZeocinTM (Invitrogen) and 2 µg/ml ponasterone A (Invitrogen) to induce protein production.

Confocal Microscopy-- Images were obtained using a four-channel Zeiss LSM 510 laser scanning confocal microscope. EGFP was excited using the 488-nm line of an argon/krypton laser. A linepass 505-nm filter was used on channel 1 for all experiments. A ×63, 1.2 numerical aperture water immersion objective was used for all experiments. Immediately before imaging, the induction medium was removed, and the cells were washed with 150 µl of 1 mM HEPES buffer, pH 7.4, containing 2.5 mM MgCl2, 1 mM NaCl, 0.6 mM KCl, 0.67 mM D-glucose, 6.4 mM sucrose and then overlaid with the same buffer. For p47phox-PX translocation measurements, PtdIns(3,4)P2 was delivered to HEK293 cells by the lipid-shuttle method using histone or neomycin as a lipid carrier as described (36). Briefly, PtdIns(3,4)P2 (20 µM) and histone (or neomycin) (20 µM) were incubated together for 10 min at 25 °C and then sonicated for 2 min in a water bath sonicator. Translocation was initiated by treating cells with the lipid-shuttle complex, and the translocation of p47phox-PX was monitored by scanning cells every 5-10 s. Treatment of cells with histone or PtdIns(3,4)P2 alone did not induce translocation of p47phox-PX after 20 min. In control experiments, cells were treated with 100 nM wortmannin for 30 min to determine the effects of PI 3-kinase on PX domain subcellular localization.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Effects of Phosphoinositides on the Monolayer Penetration of PX Domains-- Despite the availability of crystal (7, 12) and solution (37, 38) structures of several PX domains, exact roles of PIs in the membrane binding of PX domains have yet to be elucidated. Recently, it was shown that binding of PtdIns(3)P to the FYVE domains of Vps27p and Hrs induces their membrane penetration by causing changes in their local conformations and electrostatic potentials (30). To determine whether or not PIs play similar roles in the membrane binding of p40phox-PX and p47phox-PX, we measured the interactions of the PX domains with lipid monolayers of different compositions.

We first measured the effects of PtdIns(3)P on the monolayer penetration of the p40phox-PX. Specifically, a POPC/POPE (80:20) or a POPC/POPE/PtdIns(3)P (77:20:3) monolayer of a given pi 0 was spread at constant surface area, and the Delta pi was monitored after the injection of the p40phox-PX into the subphase. As shown in Fig. 1A, the p40phox-PX has low penetrating power to a POPC/POPE (80:20) monolayer with a pi c value around 20 dyne/cm, implying that the p40phox-PX has low intrinsic membrane-penetrating capability. However, incorporation of 3 mol % PtdIns(3)P into the monolayer greatly elevated the monolayer penetrating capability of the p40phox-PX, raising the pi c value near 31 dyne/cm. It has been shown that the global cellular concentration of some PIs can reach up to 3 mol % and that their local concentrations can be much higher than global concentrations (39). This suggests that PtdIns(3)P might induce the partial penetration of the p40phox-PX into cell membranes, since the surface pressure of cell membranes has been estimated to be near 31 dyne/cm (40-42). The specific nature of the PtdIns(3)P effect was demonstrated by the lack of effect by either a stereoisomer of PtdIns(3)P, phosphatidylinositol 5-phosphate, or a bulk anionic phospholipid, PtdSer, on monolayer penetration of the p40phox-PX (Fig. 1A).


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Fig. 1.   Delta pi versus pi o plots for p40phox-PX and p47phox-PX. A, p40phox-PX was allowed to interact with POPC/POPE (80:20) (open circle ), POPC/POPE/PtdIns(3)P (77:20:3) (), POPC/POPE/PtdIns(5)P (77:20:3) (), POPC/POPE/POPS (50:20:30) (triangle ), and POPC/POPE/POPS/PtdIns(3)P (62:20:15:3) (black-triangle). B, p47phox-PX was allowed to interact with POPC/POPE (80:20) (open circle ), POPC/POPE/PtdIns(3,4)P2 (77:20:3) (), POPC/POPE/POPA (77:20:3) (black-square), and POPC/POPE/POPS (50:20:30) (black-diamond ). The subphase was 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all measurements.

Our recent study showed that the p47phox-PX has a PtdIns(3,4)P2-specific site as well as a secondary site that nonspecifically bind anionic phospholipids with smaller headgroups, including PtdOH and PtdSer (12). This would suggest that membrane penetration of the p47phox-PX might be influenced by anionic phospholipids other than PtdIns(3,4)P2. To test this notion, we measured the penetration of the p47phox-PX into monolayers of various lipid compositions. We first measured the monolayer penetration of p47phox-PX to a POPC/POPE (80:20) monolayer. When compared with the p40phox-PX, the p47phox-PX showed a higher degree of penetration into the POPC/POPE (8:2) monolayer (i.e. pi c approx  28 dyne/cm) (see Fig. 1B), suggesting that it has higher intrinsic membrane penetration activity. As was the case with p40phox-PX, 3 mol % of PtdIns(3,4)P2 in the POPC/POPE monolayer greatly enhanced the monolayer penetration of p47phox-PX, increasing the pi c value to 37 dyne/cm, indicating that PtdIns(3,4)P2 would also enable the p47phox-PX to penetrate cell membranes. We then monitored the penetration of p47phox-PX into monolayers containing 3 mol % POPA or 30 mol % POPS (note that the secondary site has higher affinity for PtdOH than for PtdSer) (12). As shown in Fig. 1B, both 3 mol % POPA and 30 mol % POPS allow p47phox-PX to have pi c values above 31 dyne/cm, suggesting that these anionic phospholipids can induce the membrane penetration of p47phox-PX, presumably through interactions with the secondary lipid binding pocket (see below). This is in sharp contrast with p40phox-PX, the monolayer penetration of which was not affected by 30 mol % POPS under the same conditions (see Fig. 1A).

Effects of Mutations on the Monolayer Penetration of PX Domains-- To better understand the mechanisms by which PIs and other anionic phospholipids induce the membrane penetration of p40phox-PX and p47phox-PX, we measured the monolayer penetration of selected mutants of the two PX domains. For p40phox-PX, we mutated arginine residues (Arg58, Arg60, and Arg105) essential for specific recognition of PtdIns(3)P (7) and hydrophobic residues (Phe35, Tyr94, and Val95) in the loop regions surrounding the PtdIns(3)P binding pocket. All three arginine mutants (R58A, R60A, and R105A) exhibited reduced penetration into the POPC/POPE/PtdIns(3)P (77:20:3) monolayer (Fig. 2A), supporting the notion that PtdIns(3)P binding specifically induces the membrane penetration of p40phox-PX. Likewise, F35A, Y94A, and V95A had low monolayer penetration both with (Fig. 2B) and without (data not shown) PtdIns(3)P in the POPC/POPE monolayer, confirming the notion that these loop regions are involved in PtdIns(3)P-induced membrane penetration. In contrast, the mutation of Leu82 located in the other end of the membrane binding surface (see Fig. 8) had little effect on the monolayer penetration, showing that this part of p40phox-PX does not participate in membrane penetration (Fig. 2B).


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Fig. 2.   Effect of mutations on the monolayer penetration of p40phox-PX. A, penetration of wild type p40phox-PX (), R58Q (black-square), R60A (black-diamond ), and R105A (black-triangle) into a POPC/POPE/PtdIns(3)P (77:20:3) monolayer was monitored as a function of pi o. B, penetration of p40phox-PX (), F35A (), L82A (open circle ), Y94A (triangle ), and V95A (diamond ) into a POPC/POPE/PtdIns(3)P (77:20:3) monolayer was monitored as a function of pi o. The subphase was 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all measurements.

For p47phox-PX, we prepared two sets of mutants in two separate lipid binding sites (see Fig. 8). First, two Arg residues (Arg43 and Arg90) involved in PtdIns(3,4)P2 binding (12) and Trp80 located in an adjacent loop were mutated to Ala. Also mutated were Arg70, which is a part of the secondary binding pocket (12), and Ile65, located in an adjacent loop. Fig. 3A shows that mutations of PtdIns(3,4)P2 binding residues have direct impact on the PtdIns(3,4)P2-induced monolayer penetration of p47phox-PX. In contrast, the mutation of Arg70 had a negligible effect under the same condition. Also, the mutation of Trp80 had a greater effect on the penetration of p47phox-PX into the POPC/POPE/PtdIns(3,4)P2 (77:20:3) monolayer than the mutation of Ile65 (Fig. 3B). These results thus suggest that PtdIns(3,4)P2 binding induces the membrane penetration of the region containing Trp80 and that the secondary lipid binding site does not significantly contribute to this event. We then measured the effects of mutations on the penetration of the p47phox-PX into non-PtdIns(3,4)P2-containing monolayers. As shown in Fig. 3C, I65A and R70Q mutations exerted much greater effects on the penetration of p47phox-PX into the POPC/POPE/POPS (50:20:30) monolayer than did W80A and R90A mutations. Similar data were obtained when the POPC/POPE/POPA (77:20:3) monolayer was used (data not shown). This indicates that the binding of an anionic phospholipid to the secondary site induces the membrane penetration of the region surrounding the site, which is independent of the PtdIns(3,4)P2-induced membrane penetration of the region surrounding the PtdIns(3,4)P2-binding site. Last, we measured the penetration of p47phox-PX mutants into the POPC/POPE (70:30) monolayer to determine the origin of high intrinsic monolayer penetrating power of the p47phox-PX (Fig. 3D). Interestingly, only I65A exhibits significantly reduced monolayer penetration, which is comparable with the penetration of the p40phox-PX into the POPC/POPE (70:30) monolayer. This indicates that Ile65 is responsible for the high intrinsic monolayer penetrating activity of the p47phox-PX, which is also consistent with the above finding that the I65A mutation significantly impairs the monolayer penetration of p47phox-PX regardless of the lipid composition of monolayers.


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Fig. 3.   Effect of p47phox-PX mutations on the monolayer penetration of p47phox-PX to PtdIns(3,4)P2 and POPS containing monolayers. A, penetration of p47phox-PX (), R43Q (black-square), R70Q (black-diamond ), and R90A (black-triangle) into a POPC/POPE/PtdIns(3,4)P2 (77:20:3) monolayer was monitored as a function of pi o. B, penetration of p47phox-PX (), I65A (triangle ), and W80A () into a POPC/POPE/PtdIns(3,4)P2 (77:20:3) monolayer was monitored as a function of pi o. C, penetration of p47phox-PX (), I65A (triangle ), R70Q (diamond ), W80A (), and R90A (black-triangle) into a POPC/POPE/POPS (50:20:30) monolayer was monitored as a function of pi o. D, penetration of p47phox-PX (), I65A (triangle ), R70Q (diamond ), W80A (open circle ), and R90A (black-triangle) into a POPC/POPE (80:20) monolayer was monitored as a function of pi o. The subphase consisted of 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all measurements.

Monolayer Penetration of Full-length p40phox and p47phox-- Recently, it was shown that the C-terminal Src homology 3 (SH3) domain of p47phox interacts with the PXXP motif within the p47phox PX domain to block the PtdIns(3,4)P2-dependent membrane binding of p47phox (12). It was also shown that the phosphorylation of p47phox on up to nine sites in the C-terminal tail, including Ser303, Ser304, Ser328, Ser359, and Ser370, untethers the SH3-PX interaction to facilitate the PtdIns(3,4)P2-dependent membrane binding of p47phox (12). To determine the effects of the SH3-PX interaction and the phosphorylation on the membrane penetration of p47phox-PX, we first measured the monolayer penetration of the full-length p47phox. As illustrated in Fig. 4A, the full-length protein showed greatly reduced monolayer penetration (pi c = 28 dyne/cm) compared with the p47phox-PX under the same conditions, suggesting that the C-terminal SH3 domain interferes with the PtdIns(3,4)P2-dependent monolayer penetration of the p47phox-PX. To further test this notion, we measured the monolayer penetration of the full-length protein carrying the W263R mutation, which was shown to disrupt the SH3-PX interaction (37). Fig. 4A illustrates that this mutant has a monolayer penetration behavior, which is comparable with that of the p47phox-PX. We then prepared a quintuple mutant, S303E/S304E/S328E/S359E/S370E to examine the effects of phosphorylation of the C-terminal tail on the PtdIns(3,4)P2-dependent monolayer penetration of the full-length p47phox. Again, the monolayer penetration behavior (i.e. pi c = 36 dyne/cm) of this phosphorylation mimic mutant (see Fig. 4A) resembles that of p47phox-PX, suggesting that the phosphorylation unmasks the PX domain and thereby leads to its penetration into PtdIns(3,4)P2-containing membranes.


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Fig. 4.   Effect of initial surface pressure of monolayer on penetration of p47phox, p47phox mutants and p40phox. A, penetration of full-length p47phox (black-square), p47phox-PX (open circle ), W263R (black-diamond ), R43Q/W263R (black-triangle), and 5SE p47phox (black-down-triangle ) into a POPC/POPE/PtdIns(3,4)P2 monolayer (77:20:3) was monitored as a function of pi o. B, penetration of full-length p40phox (black-triangle) and p40phox-PX () into a POPC/POPE/PtdIns(3)P monolayer (77:20:3) was monitored as a function of pi o. p40phox (triangle ) and p40phox-PX (open circle ) were also allowed to interact with a POPC/POPE (80:20) monolayer. The subphase consisted of 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all measurements.

It has been shown that the SH3 domain of p40phox does not interfere with soluble PtdIns(3)P binding (7); however, it is not known whether or not the SH3 domain hinders membrane binding of the p40phox-PX. To answer this question, we measured the monolayer penetration of full-length p40phox. As shown in Fig. 4B, the full-length p40phox penetrated the POPC/POPE/PtdIns(3)P (77:20:2) monolayer as well as the p40phox-PX, indicating that the SH3 domain of p40phox does not interfere with the membrane binding of its PX domain.

SPR Membrane Binding Analysis of PX Domains-- We previously reported that PtdIns(3,4)P2 enhances the membrane affinity (Kd) of p47phox-PX mainly by reducing the membrane dissociation rate constant (kd), whereas PtdOH or PtdSer increases the membrane affinity by affecting both ka and kd (12). To determine the effects of PtdIns(3)P on membrane binding of p40phox-PX, we varied the concentration of PtdIns(3)P in POPC/POPE/PtdIns(3)P (80 - x:20:x) mixed vesicles immobilized to the sensor surface and measured the kinetics of membrane binding by SPR analysis. Fig. 5A shows representative sensorgrams for p40phox-PX binding to POPC/POPE/PtdIns(3)P vesicles (77:20:3), from which ka and kd values were calculated. Separate equilibrium SPR measurements yielded the binding isotherms, as shown for binding of p40phox-PX to POPC/POPE/PtdIns(3)P vesicles (77:20:3) in Fig. 5B, from which Kd values were calculated. Typically, Kd values determined from kinetic measurements (e.g. Kd = 1.3 ± 0.5 nM from Fig. 5A) using the relationship, Kd kd/ka, agree well with Kd values determined from equilibrium measurements (e.g. Kd = 0.71 ± 0.02 nM from Fig. 5B). For this reason, kinetically determined Kd values are used for evaluating the relative affinity of wild type and mutants (see Table I).


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Fig. 5.   Kinetic and equilibrium SPR measurements for binding of p40phox-PX to POPC/POPE/PtdIns(3,4)P2 (77:20:3) vesicles. A, sensorgrams from kinetic measurements. The p40phox-PX was injected at 60 µl/min at varying concentrations (0.5, 1, 2, 5, and 10 nM; see the labels). Solid lines represent the best-fit theoretical curves. B, equilibrium SPR measurements. p40phox-PX was injected at 2 µl/min at varying concentrations (0.5, 1, 2, 5, and 10 nM; from bottom to top) and Req values were measured (see inset). A binding isotherm was then generated from the Req versus [p40phox-PX] plot. A solid line represents a theoretical curve constructed from Rmax (75 ± 1.2) and Kd (0.71 ± 0.02 nM) values determined by nonlinear least-squares analysis of the isotherm using an equation: Req = Rmax/(1 + Kd/C). 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl was used for both measurements.


                              
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Table I
Binding parameters for p40phax-PX and mutants determined from SPR analysis
Values represent the mean and S. D. from three determinations. All measurements were performed in 10 mM HEPES, pH 7.4, containing 0.1 M KCl.

In the absence of PtdIns(3)P, the p40phox-PX had extremely low affinity for POPC/POPE (80:20) vesicles (Kd > 50 µM). This is consistent with our finding that the p40phox-PX has extremely low affinity for POPC/POPE (80:20) monolayer above 30 dyne/cm, which represents the surface pressure value of the large vesicles used in the SPR measurements (42). As summarized in Table I, PtdIns(3)P greatly enhanced the membrane affinity of p40phox PX; an increase in PtdIns(3)P concentration from 0.2 to 5 mol % resulted in a >700-fold decrease in Kd. As was the case with the p47phox-PX, the enhanced affinity by PtdIns(3)P was mainly attributed to reduced kd; ka increased only 1.6-fold, whereas kd decreased 112-fold when the PtdIns(3)P concentration was varied from 0.2 to 5 mol % in the vesicles. This finding is also consistent with our monolayer data indicating that PtdIns(3)P facilitates the membrane penetration of p40phox-PX, thereby allowing the domain to attain a longer membrane residence time.

The effects of mutations on binding of p40phox-PX to POPC/POPE/PtdIns(3)P (77:20:3) vesicles are also summarized in Table I. Mutation of Arg58, which was shown to be essential for interaction with the 3-phosphate group (7), caused the most dramatic 177-fold reduction in affinity, which was attributed to a reduction in ka and an increase in kd. Also, mutation of Arg105 that forms hydrogen bonds with the 4- and 5-hydroxyl groups (7) reduced membrane binding by 35-fold, and this reduced affinity was due to a reduced ka and an increased kd. Similarly, mutation of Arg60, which interacts with the 1-phosphate of PtdIns(3)P (7), resulted in an 18-fold decrease in Kd by changing both ka and kd. Since PtdIns(3)P increases the membrane affinity of the p40phox-PX by decreasing kd (see above), these results suggest that the basic residues in the binding pocket might be involved in more than PtdIns(3)P coordination (see below). On the other hand, mutations of hydrophobic residues in the loop regions (F35A, Y94A, and V95A) reduced membrane affinity of p40phox-PX by 33-, 27-, and 5.9-fold, respectively, which was primarily due to increases in kd. This is consistent with our monolayer data indicating that these residues are involved in membrane penetration. Last, L82A behaved like wild type, confirming that the region near this hydrophobic residue is not directly involved in membrane interaction.

Calculation of the Electrostatic Potential-- Our monolayer data demonstrate distinct membrane binding properties of the two PX domains (i.e. PtdIns(3)P specifically triggers the monolayer penetration of p40phox-PX, whereas PtdIns(3,4)P2 and other anionic lipids can induce the monolayer penetration of p47phox-PX). Furthermore, the p47phox-PX has higher intrinsic monolayer penetration activity than p40phox-PX in the absence of PtdIns(3,4)P2 and/or other anionic lipids. To account for these differences, we calculated the electrostatic potentials for p40phox-PX and p47phox-PX in the absence and presence of PtdIns(3)P (for p40phox-PX) and of PtdIns(3,4)P2 and PtdSer (for p47phox-PX). The results are illustrated in Fig. 6. In the absence of PtdIns(3)P, the loop regions surrounding the PtdIns(3)P binding pocket of p40phox-PX that interact with the membrane have strong positive electrostatic potential (see Fig. 6A). This positive potential is largely due to basic residues in the PtdIns(3)P binding pocket (i.e. Arg58, Arg60, and Arg105) and may initially contribute to recruiting the domain to the anionic membrane surface through nonspecific electrostatic attraction, which then leads to productive PtdIns(3)P binding. This is consistent with the finding that mutations of the basic residues in the PtdIns(3)P binding pocket reduce ka (due to reduced nonspecific electrostatic interactions) as well as increase kd (due to lower PtdIns(3)P binding) (Table I). However, this region of high positive potential also surrounds the hydrophobic residues (i.e. Phe35, Tyr94, and Val95) and is thus expected to produce an energetic barrier opposing the penetration of these residues into the low dielectric membrane interface. This is because the desolvation that is prerequisite for their membrane penetration would disrupt favorable interactions between water molecules and charged and polar groups on both the protein and membrane. Interestingly, the positive potential is dramatically reduced when PtdIns(3)P binds to the domain (see Fig. 6B). Fig. 6C shows quantitatively that the electrostatic potential surrounding the hydrophobic residues are significantly less positive when PtdIns(3)P is bound. This suggests that PtdIns(3)P may function as an electrostatic switch to decrease the highly positive potential surrounding the hydrophobic residues, thereby facilitating their membrane penetration.


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Fig. 6.   Effects of anionic lipids on the electrostatic potential of p40phox-PX and p47phox-PX. The electrostatic potential for the PX domains was calculated and visualized in the program GRASP by two-dimensional equipotential contours in 0.1 M KCl. The red contours are -25 mV; magenta, -12 mV; dark blue, +25 mV; light blue, +12 mV. A, p40phox-PX in the absence of PtdIns(3)P; B, p40phox-PX in the presence of PtdIns(3)P; C, difference between B and A; D, p47phox-PX in the absence of PtdIns(3,4)P2; E, p47phox-PX in the presence of PtdIns(3,4)P2; F, E - D; G, p47phox-PX in the absence of PtdSer; H, p47phox-PX in the presence of PtdSer; I, H - G; J, p47phox-PX without ligands; K, p47phox-PX in the presence of both PtdIns(3,4)P2 and PtdSer; L, K - J. Notice that for p47phox-PX, the positive potentials in the vicinity of Trp80 are reduced by PtdIns(3,4)P2 but not by PtdSer and that the tip of Ile65 side chain has neutral electrostatic potential with and without lipids. In K, the effect of two lipids is essentially additive.

Our electrostatic potential calculations on the p47phox-PX also provide insight into the origin of the unique membrane binding properties of p47phox-PX. As shown in Fig. 6D, the tip of the Ile65 side chain is in a region of very low electrostatic potential even in the absence of PtdIns(3,4)P2 and PtdSer, which explains how this hydrophobic residue can penetrate the POPC/POPE (80:20) monolayer to some extent. In agreement with our monolayer penetration data, PtdIns(3,4)P2 reduces the electrostatic potential of the loop region containing Trp80 (Fig. 6E), which would cause favorable partitioning of this region into the monolayer due to reduced desolvation penalty associated with the process. Calculations with PtdSer (Fig. 6H) also show that the electrostatic potential of the secondary lipid binding pocket is reduced when PtdSer binds to this site, which is consistent with the ability of PtdSer to promote the monolayer penetration of p47phox-PX. Last, we calculated the reduction in electrostatic potential of p47phox-PX in the presence of both PtdIns(3,4)P2 and PtdSer. As shown in Fig. 6K, simultaneous docking of both ligands reduces the potential of p47phox-PX most dramatically, consistent with the reported synergistic effect of the two ligands.

Subcellular Localization of PX Domains-- It has been shown that the p40phox-PX is localized to PtdIns(3)P-rich endosomes in resting porcine aortic endothelial cells (7) and COS cells (19), whereas the p47phox-PX is dispersed in the cytosol of COS cells (19). To assess the importance of membrane binding residues of p40phox-PX and p47phox-PX in their cellular membrane targeting, we monitored the subcellular localization of EGFP-tagged constructs of p40phox-PX, p47phox-PX, and respective mutants in HEK293 cells. In agreement with previous reports, the p40phox-PX showed distinct endosomal localization pattern (see Fig. 7A). The prelocalization of p40phox-PX at endosomal membranes was a PtdIns(3)P-dependent process, since treatment of the cells with PI 3-kinase inhibitor wortmannin (100 nM) resulted in a homogenous distribution of the protein in the cytosol (Fig. 7B). This notion is further supported by the finding that R58Q with greatly reduced PtdIns(3)P affinity exhibited a homogenous cytosolic distribution (Fig. 7C) under the conditions in which the wild type showed the endosomal localization. Furthermore, mutations of loop residues (F35A and Y94A) also caused cytoplasmic distribution of proteins in HEK293 cells (Fig. 7, D and E), underscoring the physiological significance of the membrane penetration of these residues in subcellular targeting of the p40phox-PX domain.


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Fig. 7.   Subcellular localization of PX domains and mutants in HEK293 cells. A, p40phox-PX; B, p40phox-PX 30 min after 100 nM wortmannin treatment; C, p40phox-PX F35A; D, p40phox-PX R58Q; E, p40phox-PX Y94A; F, p47phox-PX; G, p47phox-PX after PtdIns(3,4)P2 was fed into cells; H, p47phox-PX R90A; I, p47phox-PX R70Q; J, p47phox-PX W80A. PtdIns(3,4)P2 was fed into cells for G-J. All proteins are EGFP-tagged at their C termini. The images in A and C-F were time-independent. The image in G was taken 1 min after PtdIns(3,4)P2 treatment, whereas the images in H-J were taken 20 min after PtdIns(3,4)P2 treatment.

In contrast to p40phox-PX, the p47phox-PX was evenly distributed in the cytosol of HEK293 cells (Fig. 7F). It was reported that the p47phox-PX could be localized at membrane ruffles when COS cells were stimulated with insulin-like growth factor-1 (19). In HEK293 cells, however, insulin-like growth factor-1 failed to induce the membrane translocation of the p47phox-PX. When HEK293 cells were fed with 20 µM PtdIns(3,4)P2-histone complex, the p47phox-PX translocated to the plasma membrane in less than 1 min (Fig. 7G). This translocation was a PtdIns(3,4)P2-specific process, since R90A did not migrate to the plasma membrane even after 20 min under the same conditions (Fig. 7H). Interestingly, R70Q with reduced affinity for PtdOH or PtdSer did not translocate to the plasma membrane after 20 min (Fig. 7I), suggesting that binding of PtdSer that is abundant in the inner plasma membrane to the secondary site binding also contributes significantly to the cellular membrane targeting of p47phox-PX. Last, neither W80A (Fig. 7J) nor I65A (data not shown) showed translocation under these conditions, indicating that membrane penetration of these residues is also important for the subcellular targeting of the p47phox-PX domain.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recent finding that PX domains interact with PtdIns(3)P and other PIs suggests a new mechanism by which PIs can regulate multiple cellular processes via a spectrum of PX domain-containing peripheral proteins. The first crystal structure of p40phox-PX liganded with a water-soluble PtdIns(3)P illustrated how the domain achieves the stereospecific recognition of PtdIns(3)P (7). More recently, the crystal structure of p47phox-PX revealed the presence of two lipid binding pockets, one specific for PtdIns(3,4)P2 and the other with high affinity for anionic phospholipids with smaller headgroups, such as PtdOH (12). Despite this structural information, less is known as to how various PIs affect the in vitro and cellular membrane targeting of different PX domains.

Our previous study on the FYVE domains indicated that PtdIns(3)P binding specifically induces the membrane penetration of surface hydrophobic residues of FYVE domains, presumably by causing local conformational changes of proteins and by neutralizing the positive electrostatic potential surrounding hydrophobic loop residues that interferes with the membrane penetration of domains (30). Based on these data, we proposed a membrane binding mechanism for the FYVE domain in which initial membrane adsorption of the domain driven by nonspecific electrostatic interactions between the cationic protein surface and the anionic membrane surface is followed by specific PtdIns(3)P-triggered membrane penetration of the domain. The present study on the p40phox-PX indicates that its PtdIns(3)P-mediated membrane binding follows essentially the same mechanism. Our monolayer data clearly indicate that PtdIns(3)P binding is not a consequence of but a prerequisite for the penetration of the domain into the monolayer, the packing density of which is comparable with that of cellular membranes and large unilamellar vesicles (40-42). The penetration of hydrophobic residues (Phe35, Tyr94, and Val95) located in the loops surrounding the PtdIns(3)P-binding pocket of p40phox-PX is a specific PtdIns(3)P-dependent process, since it was abrogated either by removal of PtdIns(3)P in the monolayer or by mutation of the PtdIns(3)P-coordinating residues, Arg58, Arg60, or Arg105. The electrostatic potential calculation suggests that PtdIns(3)P binding serves as a electrostatic switch that greatly reduces the positive electrostatic potential surrounding the hydrophobic residues. As is the case with the FYVE domain, this reduction in electrostatic potential is necessary for circumventing the high desolvation penalty associated with the membrane penetration of the hydrophobic residues. Neither the present study nor the previous structural study on the p40phox-PX-PtdIns(3)P complex (7) provides direct information as to whether or not PtdIns(3)P binding causes conformational changes of the p40phox-PX. Other studies have indicated, however, that binding of a ligand to a membrane targeting domain causes local conformational changes to reorient the hydrophobic residues for more productive membrane penetration (22, 29, 30, 43). Furthermore, a recent NMR study on the PX domain of Vam7p (9) revealed that residues in its putative membrane binding loops underwent large changes in chemical shift when dodecylphosphocholine micelles were added to the PX domain-dibutanoyl PtdIns(3)P complex. Thus, it is likely that PtdIns(3)P binding also induces the reorientation of hydrophobic side chains of the p40phox-PX for their better partitioning into the lipid bilayer. Our SPR data show that the PtdIns(3)P-induced membrane penetration of these residues enhances the membrane affinity of the p40phox-PX by lowering the kd value. Also, our SPR and electrostatic potential computation data suggest that clustered cationic residues (Arg58, Arg60, Lys92, and Arg105) in the PtdIns(3)P-binding pocket are involved not only in PtdIns(3)P coordination (hence a decrease in kd) but also in initial nonspecific electrostatic adsorption to the anionic membrane surface (hence an increase in ka).

As expected from the presence of two lipid binding sites, the membrane binding mechanism of the p47phox-PX is more complex than that of the p40phox-PX. Membrane binding of p47phox-PX has two salient features. First, it has higher intrinsic monolayer penetration activity than p40phox-PX. Second, not only PtdIns(3,4)P2 but also PtdOH (or PtdSer) can induce the monolayer penetration of the p47phox-PX into the monolayer, the packing density of which is comparable with that of cell membranes. The higher intrinsic membrane-penetrating activity of p47phox-PX is attributed to a single hydrophobic residue Ile65. This residue can nonspecifically penetrate the neutral monolayers to some extent, because a part of its side chain is not surrounded by the positive electrostatic potential, and therefore its membrane penetration involves a much lesser degree of desolvation. It should be noted, however, that PtdIns(3,4)P2 and PtdOH (or PtdSer) are still required for p47phox-PX to effectively interact with the monolayer or the bilayer whose lipid packing density is comparable with that of cell membranes. Thus, the physiological significance of the higher intrinsic membrane-penetrating activity of p47phox-PX is not clear. Interestingly, PtdIns(3,4)P2 and PtdOH (or PtdSer) induce the membrane penetration of hydrophobic residues located near their binding pockets, respectively; i.e. PtdIns(3,4)P2 primarily influences Trp80, and PtdOH (or PtdSer) mainly affects Ile65. This selective effect is readily accounted for by our electrostatic potential calculations, which show that PtdIns(3,4)P2 coordination by Arg43 and Arg90 dramatically reduces the positive potential near Trp80, whereas PtdSer binding lowers the electrostatic potential near Ile65. As is suggested for p40phox-PX, it is also possible that ligand binding to each pocket induces the local conformational changes, especially in the loop regions containing hydrophobic residues, Trp80 and Ile65. We previously showed that PtdIns(3,4)P2 and PtdOH (or PtdSer) synergistically increase the membrane affinity (12). Our monolayer data as well as electrostatic potential computation indicate that this synergistic effect is due in part to a dramatic reduction in electrostatic potential when both binding pockets are occupied by ligands, which in turn promotes more effective membrane penetration of hydrophobic side chains. It would seem that the simultaneous binding of PtdIns(3,4)P2 and PtdOH (or PtdSer) to the two separate sites (i.e. simultaneous membrane penetration of two hydrophobic regions) is necessary for effective membrane targeting of the p47phox-PX, since its affinity for POPC/POPE/PtdIns(3,4)P2 (77:20:3) vesicles is about 30 times lower than that of p40phox-PX for POPC/POPE/PtdIns(3)P (77:20:3) vesicles under the same conditions (see Table I). Only in the presence of both PtdIns(3,4)P2 and PtdOH (or PtdSer) in the vesicles (i.e. POPC/POPE/POPS/PtdIns(3,4)P2 (74:20:3:3)), the p47phox-PX shows an affinity comparable with that of the p40phox-PX for POPC/POPE/PtdIns(3)P (77:20:3) vesicles (see Table I).

Taken together, we propose membrane-binding modes for p40phox-PX and p47phox-PX (see Fig. 8). Both follow essentially the same sequences for membrane binding: initial membrane adsorption driven by nonspecific electrostatic interactions between the cationic protein surface and the anionic membrane surface, followed by PI-induced membrane penetration of protein. A main difference is that for p40phox-PX, hydrophobic residues near the PtdIns(3)P-binding pocket primarily participate in membrane penetration, whereas for p47phox-PX, hydrophobic residues surrounding both PtdIns(3,4)P2-binding pocket and the secondary binding site are involved.


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Fig. 8.   Proposed membrane binding modes of p47phox-PX (A) and p40phox-PX (B). The structure of membrane-bound p47phox-PX (ribbon diagram) with a PtdIns(3,4)P2 molecule (in space-filling representation) bound to its binding pocket is modeled based upon the x-ray structure of p47phox-PX (12). The structure of membrane-bound p40phox-PX-PtdIns(3)P complex is derived from the x-ray structure of the complex (7). Mutated residues are shown in red. Note that two loop regions near PtdIns(3,4)P2- and PtdOH-binding sites, respectively, are involved in membrane penetration of p47phox-PX, whereas only the loop region surrounding the PtdIns(3)P-binding pocket participates in membrane penetration of p40phox-PX.

Subcellular localization behaviors of the two PX domains and their respective mutants are generally in line with their in vitro membrane binding properties. The p40phox-PX with high affinity for PtdIns(3)P-containing vesicles migrates to PtdIns(3)P-rich endosomes when expressed in HEK293 cells. Also, the lowering cellular level of PtdIns(3)P by wortmannin treatment and mutating the 3-phosphate ligands (Arg58 and Arg105) or membrane-penetrating hydrophobic residues all abrogate the endosomal targeting of the domain. Thus, it is evident that the cellular membrane targeting of the p40phox-PX is governed, at least in part, by the forces that control the in vitro binding of the domain to lipid vesicles. As described above, the p47phox-PX in the presence of both PtdIns(3,4)P2 and PtdOH (or PtdSer) has membrane affinity comparable with that of p40phox-PX in the presence of PtdIns(3)P. This, in conjunction with the fact that all intracellular membranes contain a certain level of anionic lipids, indicates that the lack of membrane localization by the p47phox-PX in HEK293 cells is due to the low level of PtdIns(3,4)P2 in cell membranes. This notion is supported by targeting of the domain to the plasma membrane when PtdIns(3,4)P2 was fed into cells as a histone-lipid complex. Since this method has been shown to effectively deliver PIs to all intracellular membranes (36), the selective translocation of the p47phox-PX to the PtdSer-rich plasma membrane indicates that the secondary lipid-binding site might play a key role in the membrane targeting of p47phox-PX. The secondary site has higher affinity for PtdOH than for PtdSer, and, thus, the spatiotemporal dynamics of PtdOH in conjunction with that of PtdIns(3,4)P2 would drive the translocation of p47phox-PX to the plasma membrane and other membranes, including phagocytic membranes. Our data also show that membrane penetration of hydrophobic residues surrounding the two lipid binding sites is important for subcellular localization of p47phox-PX. It was recently reported that the p47phox-PX could be localized in membrane ruffles of COS cells in response to insulin-like growth factor-1 in a PtdIns(3,4)P2-independent manner (19). However, the driving force for this translocation was not elucidated, and the same kind of translocation was not observed in HEK293 cells. The discrepancy might be simply due to lack of the specific receptor for insulin-like growth factor-1 in HEK293 cells. Further studies are necessary to fully understand the origin of this putative PtdIns(3,4)P2-independent membrane translocation of p47phox-PX.

Many PX domains contain a proline-rich, (R/K)XXPXXP sequence that is characteristic of SH3 domain binding motifs, prompting speculation that they might be interacting partners of SH3 domain proteins (1). Indeed, the PX domain of p47phox has been shown to interact with its C-terminal SH3 domain (37), which inhibits its membrane interactions (12). Our monolayer penetration measurements demonstrate that the membrane penetration of the PX domain in the full-length p47phox is severely interfered with by other parts of the protein. Dramatic effects of W263R and quintuple phosphorylation mimic mutations on the monolayer penetration of the full-length p47phox demonstrate that the C-terminal SH3 domain blocks the PtdIns(3,4)P2 (and PtdOH)-dependent membrane penetration of the PX domain and that the phosphorylation of C-terminal tail relieves this interdomain tethering. This suggests that the full-length p47phox might not spontaneously respond to the spatiotemporal dynamics of PtdIns(3,4)P2. In contrast, the SH3 domain of p40phox plays no role in inhibiting the PtdIns(3)P-dependent membrane penetration of p40phox-PX. Thus, it is expected that the full-length p40phox, when free in cytoplasm, will spontaneously respond to the spatiotemporal dynamics of PtdIns(3)P in the membranes, phagocytic membranes in neutrophils and macrophages in particular.

In summary, the present investigation demonstrates that two different PIs, PtdIns (3)P and PtdIns(3,4)P2, have a similar effect on membrane binding for two PX domains. This, in conjunction with our previous finding that PtdIns(3)P has essentially the same effect on membrane binding of structurally distinct FYVE domains, suggests that PI-induced membrane penetration is a common mechanism for many membrane targeting domains. PX domains have broad PI specificities and variable amino acid sequences around their putative PI-binding pockets. Thus, this work would serve as a framework with which one can comprehensively study the roles of PIs in the membrane targeting of a wide spectrum of PX domains.

    ACKNOWLEDGEMENTS

We thank Drs. Roger L. Williams and Olga Perisic for kindly providing the expression vectors for p40phox, p47phox, p40phox-PX, p47phox-PX, and several mutants.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM66147 (to D. M.), GM57568 (to K. S. B.), and GM52598 and GM53987 (to W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois at Chicago, 845 W. Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho@uic.edu.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212579200

    ABBREVIATIONS

The abbreviations used are: PI, phosphoinositide; EGFP, enhanced green fluorescent protein; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; POPA, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoehthanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; SH3, Src homology 3; SPR, surface plasmon resonance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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