atypical membrane-embedded phosphatidylinositol 3,4

Atypical Membrane-embedded Phosphatidylinositol3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47phox PhoxHomology (PX) Domain Revealed by NMR*□S

Received for publication, December 11, 2011, and in revised form, March 18, 2012 Published, JBC Papers in Press, April 4, 2012, DOI 10.1074/jbc.M111.332874

Pavlos Stampoulis‡§, Takumi Ueda‡, Masahiko Matsumoto‡, Hiroaki Terasawa‡, Kei Miyano¶, Hideki Sumimoto¶,and Ichio Shimada‡�1

From the ‡Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, the §Japan Biological InformaticsConsortium, Tokyo 104-0032, the ¶Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, and the�Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology,Tokyo 135-0064, Japan

Background: The p47phox PX domain binds PI(3,4)P2 during assembly and activation of NADPH oxidase.Results: The PI(3,4)P2-binding site on the p47phox PX domain was identified by NMR.Conclusion: The position of the phosphoinositide-binding site on p47phox PX is unique.Significance: Our results provide insight into a particular role of p47phox PX in the assembly and activation of phagocyteNADPH oxidase.

The Phox homology (PX) domain is a functional module thattargets membranes through specific interactions with phos-phoinositides. The p47phox PX domain preferably binds phos-phatidylinositol 3,4-bisphosphate (PI(3,4)P2) andplays a pivotalrole in the assembly of phagocyte NADPH oxidase.We describethe PI(3,4)P2 binding mode of the p47phox PX domain as identi-fied by a transferred cross-saturation experiment. The identi-fied PI(3,4)P2-binding site, which includes the residues ofhelices �1 and �1� and the following loop up to the distortedleft-handed PPII helix, is located at a unique position, as com-pared with the phosphoinositide-binding sites of all other PXdomains characterized thus far. Mutational analyses corrobo-rated the results of the transferred cross-saturation experi-ments. Moreover, experiments with intact cells demonstratedthe importance of this unique binding site for the function of theNADPH oxidase. The low affinity and selectivity of the atypicalphosphoinositide-binding site on the p47phox PX domain sug-gest that different types of phosphoinositides sequentially bindto the p47phox PX domain, allowing the regulation of the multi-ple events that characterize the assembly and activation of phag-ocyte NADPH oxidase.

The phagocyte NADPH oxidase is a multicomponentenzyme that produces reactive oxygen species as a defenseagainst invading microorganisms. Its importance for innateimmunity is exemplified by chronic granulomatous disease, agenetic impairment of NADPH oxidase that leads to recurrentdiseases and death (1, 2). The NADPH oxidase consists of cyto-

solic (p47phox, p40phox, p67phox, and small GTPase Rac1 orRac2) and membrane-integrated components (gp91phox andp22phox). The enzyme switches from an inactive state to anactive state and is capable of producing reactive oxygen specieswhen the cytosolic components are recruited to the membraneand interact with the membrane-integrated components toform a functional complex (3–6).p47phox and p40phox contain PX2 domains, which are crucial

for the recruitment of cytosolic components (7–10). In addi-tion, the PXdomain of p47phox is important for the activation ofthe NADPH oxidase, based on the observation that in stimu-lated cells the presence of a PX-truncated p47phoxmutant led todecreased superoxide production (10).The PX domain is a functional module that targets mem-

branes through a specific interaction with membrane-embed-ded phosphoinositides (11, 12). Although the p40phox PXdomain specifically binds to PI3P, the p47phox PX domainreportedly binds to PI(3,4)P2, PA, and PtdSer.

The crystal structure of p40phox in complex with C4-PI3P hasbeen solved. In the structure, Arg-60 interacts with the 3-phos-phate (through its backbone) and the inositol 2-hydroxyl and1-phosphate groups (through its side chain) of the bound PI3P.However, Lys-92 interacts strongly with the 1-phosphate groupof PI3P. The three-dimensional structures of p47phox PX alonehave been determined by solution NMR (13) and x-ray crystal-lography (14). However, the structure of p47phox PX in complexwith its ligand has not been reported. The crystal structure of itsfree form revealed two sulfate molecules bound in two distinctsites (14).One of the sulfates is hydrogen-bonded toArg-43 and

* This work was supported by a grant from the Japan New Energy and Indus-trial Development Organization and the Ministry of Economy, Trade, andIndustry.

□S This article contains supplemental Figs. S1–S10 and additional references.1 To whom correspondence should be addressed: Graduate School of Phar-

maceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel./Fax: 81-3-3815-6540; E-mail: [email protected].

2 The abbreviations used are: PX, Phox homology; TCS, transferred cross-sat-uration; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; CPM, 7-diethyl-amino-3-(4�-maleimidylphenyl)-4-methylcoumarin; IAEDANS, 5-((((2-iodo-acetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; PA, phospha-tidic acid; PtdSer, phosphatidylserine; POPC, 1-palmitoyl-2-oleoyl-L-phos-phatidylcholine; HSQC, heteronuclear single quantum coherence; POPE,1-palmitoyl-2-oleoyl-phosphatidylethanolamine; PI3P, phosphatidylinosi-tol 3-phosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 21, pp. 17848 –17859, May 18, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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Thr-45 on the C-terminal end of the �3 strand, and it coincideswith the 3-phosphate of the bound PI3P in the crystal structureof p40phox PX. The second sulfate is hydrogen-bonded toHis-51 andLys-55 on the�1 helix and toArg-70 on the adjacentloop that leads to the distorted left-handed PPII helix. Based onthese results and surface plasmon resonance-based mutationalanalyses, the first sulfate-binding site was proposed to be thePI(3,4)P2-binding site, and the second sulfate-binding site wasconsidered to be the PA-binding site. In the crystal structure ofthe CISK PX domain, however, one of the two bound sulfategroupswas found in a completely unexpected area, which prob-ably represents a nonspecific binding site (15). Therefore, it isunclear whether these sulfate-binding sites of p47phox PX arethe actual ligand-binding sites, and thus the mechanism of thebroad ligand specificity of p47phox PX is still unclear.

Under physiological conditions, phosphoinositides interactwith effector proteins in the context of lipid bilayers. In somecases, the consideration of membrane-mimicking conditionscan greatly affect the results of in vitro studies. For instance, theability of the actin regulatory protein N-WASP to sense phos-phatidylinositol 4,5-bisphosphate density in the membrane islost when soluble phosphatidylinositol 4,5-bisphosphate orinositol trisphosphate headgroups are used instead (16). Asanother example, the actual affinity of the vacuole protein sort-ing HOPS for PI3P can be revealed only by a liposome assay(17).NMR is a useful technique for investigations of biological

systems within a certain size limit (currently in the area of 800kDa) (18). To overcome this inherent size limitation in NMR,appropriate NMR methodologies and sample preparations arenecessary. This is particularly true in cases involving biomem-branes, because a protein bound on a lipid bilayer is a hugebiological structure that cannot be observed easily by NMR.Transferred cross-saturation (TCS) is an NMR method thatenables the identification of the residues of protein ligands inclose proximity to huge (�100 kDa) complexes (19–21). Suc-cessful applications of TCS for huge and/or inhomogeneoussystems have previously been reported (22–29).In this study, we employed TCS experiments to identify the

PI(3,4)P2-containing membrane-interacting interface on thep47phox PX domain, using liposomes as a membrane-mimick-ing environment. Mutational analyses verified the results ofTCS and revealed that Lys-55 is important for PI(3,4)P2 recog-nition.Moreover, we demonstrate that Lys-55 is also importantfor the proper function of phagocyte NADPH oxidase. Alto-gether, our data indicate that the p47phox PX domain bindsPI(3,4)P2 in a unique area, as comparedwith the phosphoinosit-ide-binding sites of all of the other PX domains characterizedthus far (30–35).

EXPERIMENTAL PROCEDURES

Sample Preparation—The human p47phox PX domain (resi-dues 1–128), encoded on the pGEX-2T vector (GEHealthcare),was expressed and purified as reported (13). The humanp40phox PX domain (residues 1–167) on the pGEX-2T vectorwas expressed and purified similarly. The DNA encoding theVam7p PX domain (residues 1–122) was prepared by total syn-thesis using the ligation-independent cloning method. The

Vam7p PX on the pET43a vector was expressed as an untaggedprotein and purified by a combination of anion and cationexchange chromatography. Mutants were prepared accordingto the QuikChange (Stratagene) protocol, and their expressionand purification were performed similarly. Their correct fold-ing was confirmed by an inspection of their HSQC spectra. Theuniformly double-labeled 2H-15N PX domain was prepared bygrowing cells in 99% D2O minimal medium, with 1 g liter�1

15NH4Cl and 3 g liter�1 97% [2H]glucose. The cells were grownat 37 °C to an A600 of 1.0 and then were induced by 1 mM iso-propyl 1-thio-�-D-galactopyranoside and incubated overnightat 25 °C. The cells were harvested and lysed by sonication, andthe supernatant was applied to glutathione-Sepharose 4B beads(GE Healthcare). The GST tag was removed by thrombin, andthe isolated 2H-15N PX domain was purified according to thereported procedure (13). Large unilamellar vesicles were pre-pared by extrusion. Briefly, the required amounts of POPC(Avanti), POPE (Avanti), and di-C16PI(3,4)P2 (Echelon),ordi-C16PI3P (Echelon) were mixed in CHCl3, dried over a N2stream, and placed under vacuum overnight. The lipid mixturewas resuspended in 5 mM [2H]MES, pH 5.5, 90% D2O, or 5 mM

MES, pH 5.5, 100% H2O at 37 °C. Hydrated lipids were sub-jected to extrusion using an Avanti mini-extruder with a100-nm polycarbonate filter. Phospholipid concentrationswere determined by a phosphate assay (36). The size of theprepared liposomeswas estimated byDynamic Light Scatteringexperiments, using a DynaPro instrument at 25 °C with 10%laser intensity. The phosphoinositide-containing and controlliposomes gave an average radius of 60.4 and 65.9 nm, respec-tively, with high homogeneity (low % polydispersity, 100%intensity, and 100% mass). The lamellarity of prepared lipo-somes was calculated as reported previously (37), and for thephosphoinositide-containing and control liposomes, the lamel-larities were estimated to be 0.8 and 0.72, respectively. Thestability of the liposomes during the interactions with proteinswas evaluated by Dynamic Light Scattering experiments, inwhich the size of the liposomes was recorded over time. Nosignificant change was observed in the size of the liposomes.The stability of the liposomes was further evaluated by fluores-cence measurements of liposomes loaded with carboxyfluores-cein. No significant change in self-quenching of carboxyfluo-rescein was observed over time. For titration experiments,stock solutions of di-C4PI(3,4)P2 (Echelon), di-C6PA (Avanti),and di-C6PtdSer (Avanti) were prepared in 5 mM MES, 5 mM

DTT, pH 5.5. Phosphates (Wako) were mixed to produce aphosphate buffer at pH 5.5.Fluorescence Assay—Cys-less p47phox PX (Cys-98 and Cys-

111were substitutedwithAla and Ser, respectively) was labeledat position 21 (CPM-(21)p47phox PX) or alternatively at posi-tion 65 (CPM-(65)p47phoxPX)with the fluorescent probeCPM,after the introduction of the required Cys in the labeling site.For labeling, a 10-fold molar excess of CPMwas used, in 50mM

Tris, pH 7.4, and 100 mMNaCl, at 4 °C overnight. The reactionmixture was cleared by centrifugation, and the excess CPMwasremoved by passage through a PD-10 column. The purity ofCPM-labeled PX was assessed by comparison of a CoomassieBrilliant Blue-stained gel and aUV-transilluminated SDS-poly-acrylamide gel for the same sample. The labeling efficiency was

Binding of Liposome-embedded PI(3,4)P2 on p47phox PX Domain

MAY 18, 2012 • VOLUME 287 • NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 17849


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calculated from a molar extinction coefficient of 33,000 (M�1

cm�1) for CPM, and it was �90%. The correct folding of CPM-labeled p47phoxPX and its ability to interact with C4-PI(3,4)P2were confirmed by an inspection of its HSQC spectra with andwithout ligand. For the 1,5-IAEDANS labeling of Cys-lessp47phox PX at position 21 or position 80, a 20-fold molar excessof 1,5-IAEDANS was used in 20 mM Tris, 100 mM KCl, pH 7.5,at 20 °C, overnight and gave �90% labeled product. The stocksolution of lipid vesicles and the sample solution of labeledp47phox PXwere prepared in 5mMMES, pH 5.5.Measurementswere conducted on an RF-5300PC Shimadzu spectrofluoro-photometer. The CPM-labeled sample was excited at 384 nm,and the emission was detected at 470 nm at 22 °C. TheIAEDANS-labeled sample was excited at 336 nm, and the emis-sion was detected at 490 nm at 22 °C.NMR Spectroscopy—HSQC spectra were recorded on a

Bruker Avance 500 or Bruker Avance 600 spectrometerequippedwith a cryo-probe. Assignment data were provided byDr. Hidekazu Hiroaki, Kobe University. We reconfirmed themby a combination of three-dimensional NMR spectra and selec-tive labeling. The backbone NMR assignment data of p40phoxPX and T45R/P78K/K55Emutant of p47phox PX were obtainedby standard three-dimensional NMR measurements. The pre-viously reportedNMRassignment data of Vam7pPXwere used(38). For the determination of the lamellarity of prepared lipo-somes, the one-dimensional 31P NMR spectra were recordedon a Bruker DRX 400 spectrometer. Unless otherwise stated,measurements were conducted at 25 °C, with NMR samplesprepared in 5 mM MES, 5 mM DTT, pH 5.5. For TCS experi-ments, NMR samples containing 0.1 mM 2H-15N PX domainand 2.17 mM total lipid concentration of unlabeled liposomeswere prepared in 5 mM [2H]MES, [2H]DTT, pH 5.5, 90% D2O.The TCS experiment was performed as described previously(19, 22). Selective saturation of the proton content of the lipo-some components was achieved by using theWURST-2 broad-band decoupling scheme. The saturation frequency was set at1.3 ppm, and the saturation time was 1 s. All spectra were pro-cessed with the program nmrPipe, and data analysis wasaccomplished with the Sparky program (University of Califor-nia, San Francisco).Activation System for the Phagocyte NADPH Oxidase—The

cDNA for human wild type (WT) p47phox was prepared asdescribed previously (39). Amutation leading to the K55A sub-stitution in p47phox was introduced by PCR-mediated site-di-rected mutageneses. The cDNAs were ligated to a pEF-BOS-based vector for expression of p47phox (WT) and p47phox(K55A) as HA-tagged protein in mammalian cells (39). All theconstructs were sequenced for confirmation of their identities.The K562 cells stably expressing gp91phox and p67phox (K562-gp91phox/p67phox cells) were prepared as described previously(40). The cells (5 � 106 cells/ml) were electroporated in thepresence of 60 �g of pEF-BOS-HA-p47phox (WT) or pEF-BOS-HA-p47phox (K55A) at 170 V and 1,070 microfarads using aGenePulser (Bio-Rad). After culture for 48 h, the superoxide-producing activity of these transfected cells was determined bysuperoxide dismutase-inhibitable chemiluminescence withan enhancer-containing luminol-based detection system(DIOGENES, National Diagnostics), as described previously

(39, 41). After the addition of DIOGENES, cells (1.0� 106 cells/ml) were preincubated for 5 min at 37 °C and subsequentlystimulated with 200 ng/ml phorbol 12-myristate 13-acetate.The chemiluminescence was monitored at 37 °C using a lumi-nometer (Auto Lumat LB953; EG&G Berthold). For estimationof protein levels of HA-p47phox, p67phox, and p22phox, celllysates of K562 cells were subjected to SDS-PAGE, transferredto a polyvinylidene difluoride membrane (Millipore), andprobed with an anti-HA monoclonal antibody (CovanceResearch Products), an anti-p67phox monoclonal antibody (BDBiosciences), or anti-p22phox polyclonal antibodies (Santa CruzBiotechnology). The blots were developed using ECL-prime(GEHealthcare) for visualization of the antibodies, as describedpreviously (41).

RESULTS

Determination of Affinity of the Complex between the p47phoxPX Domain and Insoluble Long Lipid Chain, C16-PI(3,4)P2,Embedded in Liposomes—For the quantitative analyses of theaffinity of the p47phoxPXdomain for insoluble long lipid chains,C16-PI(3,4)P2 embedded in liposomes, a 7-diethylamino-3-(4�-maleimidylphenyl)-4-methylcoumarin (CPM)-based fluores-cence assay (16, 42) was conducted. The increase of fluores-cence of the CPM-labeled PX domain was observed upon theaddition of C16-PI(3,4)P2-containing liposomes, whereas theCPM-labeled PX domain showed no interaction in an identicalexperiment, using control liposomes without PI(3,4)P2 (com-posed of POPC/POPE � 80:20) (Fig. 1). Based on the fluores-cence titration curve, a dissociation constant value of 24.8 �M

FIGURE 1. CPM-based fluorescence assay for Kd estimation. Binding iso-therms obtained by titrating lipid vesicles (large unilamellar vesicles) com-posed of POPC/POPE/C16-PI(3,4)P2 � 79.5:20:0.5 (A) or control vesicles com-posed of POPC/POPE � 79.5:20 (B), into a solution of 0.3 �M CPM-(21)p47phox

PX in 5 mM MES, pH 5.5. The sample was excited at 384 nm, and the emissionwas detected at 470 nm at 22 °C. Data were fit by the nonlinear least squaresmethods with the Microcal Origin software, using the reported equation (53).In the titration with control vesicles, the x axis corresponds to the lipid con-centration (in �M), as measured for a hypothetical 0.5% component of controlliposomes. The error bars in A represent average � 1 S.D. calculated fromthree independent experiments.




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was obtained, which was similar to the value previouslyreported from surface plasmon resonance analyses, using lipo-somes composed of POPC/POPE/PtdIns(3,4)P2 � (79.5:20:0.5)(see Table 3 of Ref. 14) or liposomes of increased percentage ofPtdIns(3,4)P2 (43).Interaction of p47phox PX Domain with an Insoluble Long

Lipid Chain, C16-PI(3,4)P2, Embedded in Liposomes—In thisstudy, we performed TCS experiments to investigate the inter-actions of C16-PI(3,4)P2-containing liposomes with the p47phoxPX domain to identify the C16-PI(3,4)P2-containing mem-brane-interacting interface on the p47phoxPXdomain.Anover-view of the experiment is shown in Fig. 2A. The TCS experi-ments are performed with an excess amount of the[U-2H,15N]p47phox PX domain, relative to the C16-PI(3,4)P2embedded in the liposomes. In the TCS experiment, the com-plex is irradiated at a frequency centered at the aliphatic protonresonances, which exclusively affects the liposomes, because

almost no aliphatic protons exist in the deuterated PX domain.The saturation in the liposomes is transferred to all of thehydrogen atoms of the liposomes, through intramolecularcross-relaxation (spin diffusion). The saturation is not limitedwithin the liposomes but is transferred to the residues at theinterface of the PX domain bound to the liposomes, throughintermolecular cross-relaxation (cross-saturation). If the com-plexes have a sufficiently fast exchange rate between the freeand bound states, then the saturation in the interface residues iseffectively transferred to the free state of the PX domain. As aresult, the irradiation causes a reduction in the intensities of theresonances from the interface residues of the PX domain in thefree state.The efficiency of the TCS effects depends on various exper-

imental conditions, such as the binding constant betweenligands and receptors, the molar ratio of the receptors to theligands, and the molecular weight of the receptors. We investi-

FIGURE 2. TCS experiment. A, overview of the TCS experiment for analyzing the interaction between p47phox and PI(3,4)P2-containing liposomes. B, portionsof the 1H-15N HSQC spectra of uniformly labeled [2H,15N]p47phox PX domain, recorded in the presence of C16-PI(3,4)P2-containing liposomes without saturation(left) and with saturation (right). Signals with strong and moderate intensity losses are enclosed in red and orange boxes, respectively. The recorded HSQCspectra correspond to the free state of the p47phox PX domain. Assignment data were provided by Dr. Hidekazu Hiroaki (13). C, interacting interface ofC16-PI(3,4)P2-containing liposomes on the p47phox PX domain, as identified by the TCS experiment. Uniformly double-labeled [2H,15N]p47phox PX, in 5 mM

[2H]MES, 5 mM [2H]DTT, 90% D2O, pH 5.5, was incubated with large unilamellar vesicles composed of POPC/POPE/C16-PI(3,4)P2 � 79.5:20:0.5, prepared in thesame buffer. The TCS experiment was conducted in the selective saturation mode for both the aliphatic (lipid tail) and nonaliphatic (polar head) protons of theliposome components. The intensity reduction ratio (RI) was calculated as ((I0 � I)/I0)�100, where I0 represents the intensity per residue of p47phox PX withoutsaturation, and I is the intensity per residue with saturation. Residues are color-coded according to their intensity reduction, with red and orange for strong (RI�0.3) and moderate (0.25 � RI � 0.3) reduction, respectively. Asterisks mark the position of proline residues. The secondary structure (based on its crystalstructure) is shown. The error bars represent the root sum square of (noise level/signal intensity) in the two spectra with and without irradiation. D, mapping ofresidues displaying strong intensity reductions in the TCS experiment with PI(3,4)P2-containing liposomes, on a ribbon representation and on the surface of thecrystal structure of p47phox PX. The residues are color-coded as in C. The bound sulfates are shown in a stick representation. The molecular diagrams weregenerated by Web Lab Viewer Pro (Molecular Simulations, Inc.).




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gated the effects of the physical parameters on TCS by a simu-lation (44), which indicated that the TCS effects would be suf-ficiently observed under the present experimental condition(supplemental Fig. S1).To investigate whether the TCSmethod can accurately iden-

tify the membrane-interacting interface on a peripheral mem-brane protein, the method was first employed on a modelprotein, the Vam7p PX domain. The location of the phosphoi-nositide-binding site on this PX domain is well established. Theresults of the TCS experiment for the interaction of the Vam7pPX domain with C16-PI3P-containing liposomes are shown insupplemental Fig. S2. Significant intensity reductions wereselectively observed in its reported PI3P binding pocket.Subsequently, we applied the TCS experiments to identify

the C16-PI(3,4)P2-containing membrane-interacting interfaceon the p47phox PX domain. The spectra with and without irra-diation are shown in Fig. 2B. Several residues, includingGlu-49,Lys-55, and Met-57, are affected by the irradiation, althoughothers are not. As shown in Fig. 2,C andD, residues Glu-49 andPhe-58 of helix �1 and Ile-71 of the following loop displayedintensity losses within the 0.3–0.5 range. Moderate 0.25–0.3intensity losses were exhibited by Tyr-48, Lys-55, and Met-57in helix �1, as well as by Gly-63 in the following loop. All ofthese residues form a well defined area on the surface of thep47phox PX domain, outlined by helix �1, helix �1�, and the fol-lowing loop up to the distorted left-handed PPII helix. Theectopic intensity losses shown by Met-27, which is located faraway from the main affected area, might be the result ofenhanced spin diffusion, due to the large molecular weight ofthe liposomes.To investigate the intramolecular saturation effects induced

by the residual protons in the [U-2H,15N]p47phox PX domain,an identical TCS experiment, using the 2H-15N PX domainalone and without liposomes, was performed (supplementalFig. S3, A and B). As a result, no selective intensity reductionswere observed for the residues of the p47phox PX domain. Toexamine the effect of the nonspecific binding between thep47phox PX domain and POPC and/or POPE, we performed theTCS experiments, using liposomes without PI(3,4)P2 (supple-

mental Fig. S3C). As a result, no selective intensity reductionswere observed for the residues of the p47phox PX domain.Based on the above experiments, we conclude that the C16-

PI(3,4)P2-containing membrane-interacting interface on thep47phox PX domain is the area on the surface of the protein,formed by helix �1, helix �1�, and the following loop up to thePPII helix. Hereafter, this area and the site that correspond tothe PI3P-binding site of p40phox PX are referred to as “atypicalbinding site” and “typical binding site,” respectively.Interaction of p47phox PX with Soluble C4-PI(3,4)P2—To

investigate the interaction between the p47phox PX domain andthe polar headgroup of PI(3,4)P2, NMR titration experimentswith soluble phosphoinositides were performed. Chemicalshifts ofmain chain amideNH and arginineN�Hgroups, whichwere observed in these experiments, are affected by both thedirect binding effects and the conformational change in eachresidue (45). As shown in Fig. 3A and supplemental Fig. S4,soluble C4-PI(3,4)P2 induced chemical shift perturbations forspecific signals on the HSQC spectrum of the wild type p47phox

PX. Most of these perturbed signals were derived from the res-idues in the loop between helix �1 and the PPII area (Fig. 3B),which is located inside the membrane-interacting interfaceidentified by the TCS experiments (Fig. 2D), although severalresidues that are outside the membrane-interacting interfaceand within the site that corresponds to the PI3P-binding site ofp40phox PX were also perturbed. The �NH of Arg-70, whichcoordinates the sulfate within the identified membrane-inter-acting interface, displayed a strong chemical shift perturbation(Fig. 3C). From the titration curve, a Kd value of 38.7 �M wasobtained. Further structural analyses using intermolecularNOEs were hampered by the low signal intensities of the com-plex, which are probably due to exchange broadening.For the comparison of the binding modes, the titration

experiment was also performed with the model proteins thep40phox PX andVam7p PXdomains. As shown in supplementalFig. S5, the resonances from the residues in the PI3P bindingpocket of the p40phox PX and Vam7p PX domain were selec-tively shifted upon the addition of soluble C8-PI3P.

FIGURE 3. Interaction of WT-p47phox PX with C4-PI(3,4)P2. A, chemical shift perturbation profile of the p47phox PX domain caused by C4-PI(3,4)P2. Normalizedchemical shift changes were calculated based on the equation n � 25�((��H)2 (��N/5)2)1/2 (49). Residues that showed strong (N �5 ppm) and moderate (3ppm � N � 5 ppm) normalized chemical shift changes are colored red, and orange, respectively. The error bars represent 25�(��R1H ��N�R15N/52)/N, whereR1H and R15N are the digital resolutions in ppm in the 1H and 15N dimensions, respectively. The inset shows superimposed portions of the 1H-15N HSQC spectraof [U-15N]p47phox PX with (red) and without C4-PI(3,4)P2 (black). The full spectrum is shown in supplemental Fig. S4. B, mapping of residues displaying strongC4-PI(3,4)P2-induced chemical shift perturbations onto a ribbon representation of the crystal structure of p47phox PX. The residues are color-coded as in A.C, chemical shift changes for the Arg-�NH signals of p47phox PX upon interaction with C4-PI(3,4)P2. p47phox PX (0.2 mM) was incubated with no C4-PI(3,4)P2 (red)and with 0.2 mM C4-PI(3,4)P2 (blue). Peaks were assigned through mutations.




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Interaction of p47phox PX with Various Soluble Ligands—Toclarify the ligand specificity of the p47phox PX domain, we fur-ther investigated the interaction between the p47phox PXdomain and other soluble phosphoinositides. Although thep47phox PX shows a preference for PI(3,4)P2, it can also bind toother types of phosphoinositides, such as PI3P and PI(4,5)P2,with lower affinity. They induced similar pattern of chemicalshift perturbation on p47phox PX and therefore are probablyaccommodated in the same area as the PI(3,4)P2 (supplementalFig. S6).PA and PtdSer have been reported as putative interacting

partners of the p47phox PX domain (14). We assessed the bind-ing of soluble C4-PtdSer or C6-PA to the p47phox PX domain byNMR chemical shift perturbation experiments. The addition of0.5 mM PtdSer did not induce any chemical shift perturbationsof the resonances from the p47phox PX domain (Fig. 4A). How-ever, the NMR signals from the p47phox PX domain were per-turbed upon the addition of 0.5 mM C6-PA (Fig. 4A). To exam-ine whether C6-PA binds to the PI(3,4)P2-interacting area ofp47phox PX, determined by the TCS experiments, competitionexperiments were performed for the PA-p47phox PX domainand PI(3,4)P2-p47phox PX domain interactions. As a result, thechemical shifts of the resonances from the 0.2 mM p47phox PXdomain with both 0.3 mMC4-PI(3,4)P2 and 0.6 mMC6-PAweresimilar to those with 0.3mMC4-PI(3,4)P2 (Fig. 4B) but differentfrom thosewith 0.6mMC6-PA (Fig. 4B), suggesting that PA andsoluble C4-PI(3,4)P2 competitively bind to the p47phox PXdomain.To obtain further evidence, the p47phox PX was titrated with

phosphate buffer. At a low concentration, the phosphatesinduced chemical shift changes in the same area on the surfaceof p47phox PX as C4-PI(3,4)P2. In contrast, Thr-45, a residuethat coordinates the sulfate outside the identified phospho-inositide-interacting interface, showed a strong chemical shiftchange only at high phosphate concentrations, suggesting thelack of a specific interaction (supplemental Fig. S7).Mutational Analyses to Reveal the Residues Important for

Phosphoinositide Binding—To confirm the results of the NMRexperiments, a series of p47phoxPXmutants were prepared, andtheir respective affinities for soluble C4-PI(3,4)P2 were assessed

by NMR titration experiments (Fig. 5 and Table 1). Theobtained spectra and titration curves of the alanine mutants ofArg-43 and Thr-45, which formed hydrogen bonds with thesulfate outside the identified phosphoinositide interacting areaof p47phox PX in the crystal structure (Fig. 5A), were similar tothose of thewild type (Fig. 5B, supplemental Fig. S4B, andTable1). In contrast, themutations inside the phosphoinositide inter-acting area, as determined by the TCS experiments (R70K,H51Q, and H74A), reduced the affinity. Among them, the res-onances from the K55A mutant were not perturbed upon theaddition of soluble C4-PI(3,4)P2 (Fig. 5C and Table 1).

Moreover, to examine whether the K55A mutant also abol-ishes PI(3,4)P2 binding in the presence of a membrane, a CPM-based fluorescence assay was conducted. Although the fluores-cence of the CPM-labeled T45A mutant was increased uponthe addition of C16-PI(3,4)P2-containing liposomes (gray downtriangle in supplemental Fig. S8) the K55Amutant was not per-turbed (gray diamonds in supplemental Fig. S8). The fluores-cence of another mutant of Lys-55, K55E, was also not affectedby the addition of C16-PI(3,4)P2-containing liposomes (Table2). The fluorescence intensities ofK55AandK55Ewere also notperturbed upon the addition of liposomes containing C16-PI3P,C16-PI(4,5)P2, and C16-PI(3,5)P2 (data not shown).Role for Lys-55 of p47phox PX Domain in Phagocyte Oxidase

Activation—Weexamined the role of Lys-55 in the activation ofthe NADPH oxidase in intact cells (Fig. 6). K562-gp91phox/p67phox cells, which lack p47phox but contain the other essentialcomponents of the phagocyte oxidase (40), were transfectedwith cDNA for the wild type and the K55A mutant of the HA-tagged p47phox and stimulated with phorbol 12-myristate13-acetate. As a result, the amount of superoxide produced incells that express the K55A mutant was �40% less than that incells that express the wild type. We confirmed that the expres-sion level of p47phox, p67phox, and p22phox are similar in thesecells. Thus, Lys-55 would be important for the oxidaseactivation.T45R/P78K Mutations Restore the PI3P-binding Site of

p40phox PX on p47phox PX—We showed that the Lys-55mutantof p47phox PX is unable to interact with phosphoinositides.Guided by the structural similarity between p47phox PX and

124

8.5 8.0

122

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ppm

L101

A86

Y97

L54

V120

124

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122

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L101V120

Y97

A86H51

L54E96

ppm

A Bp47phox PX p47phox PX + PAp47phox PX + PtdSer

PA-bound p47phox PXPA-bound p47phox PX + PI(3,4)P2PI(3,4)P2-bound p47phox PX

1H 1H

15N 15N

FIGURE 4. Interactions between p47phox and various ligands. A, p47phox PX interacts with C6-PA but not with C4-PtdSer. Superimposed portions of the HSQCspectra of 0.1 mM p47phox PX domain alone (black) and in complex with 0.5 mM C4-PtdSer (cyan) or with 0.3 mM C6-PA (magenta) are shown. B, interaction ofC6-PA-bound p47phox PX with C4-PI(3,4)P2. C4-PI(3,4)P2 replaces C6-PA on the p47phox PX domain. Superimposed portions of the HSQC spectra of 0.2 mM p47phox

PX domain with 0.6 mM C6-PA (magenta) and with 0.6 mM C6-PA and 0.3 mM C4-PI(3,4)P2 (green) are shown. The HSQC spectrum of 0.2 mM p47phox PX domainwith 0.3 mM C4-PI(3,4)P2 is shown in red.




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p40phox PX, we attempted to establish a p47phox mutant thatmimics the p40phox PX-like ligand-binding site and ligand spec-ificity. In the crystal structure of p40phox PX in complex with

TABLE 1Values of the dissociation constants for the interaction of solubleC4-PI(3,4)P2 with WT and mutants of p47phox PX domain, obtainedfrom NMR titration curvesND indicates not detectable.

Protein Kd �2

�M

WT 39 � 12 0.044T45A 37 � 8 0.073R43A 30 � 10 0.033H51Q 141 � 27 0.012R70K 85 � 1 0.002H74A 72 � 15 0.017K55A ND

FIGURE 5. Mutational analysis of the interaction between p47phox andC4-PI(3,4)P2. A, position of inserted mutations relative to bound sulfates inthe crystal structure of p47phox PX. Arg-43 and Thr-45, which are in the typicalbinding site, are colored green, and His-51, His-55, Arg-70, and His-74, whichare in the atypical binding site, are colored blue. Thr-45 and Arg-43 werehydrogen-bonded to the sulfate residing outside the PI(3,4)P2-containingmembrane-interacting interface. B and C, C4-PI(3,4)P2-induced chemical shiftperturbation on mutants of p47phox PX. Superimposed portions of 1H-15NHSQC spectra of [U-15N]p47phox PX with (red) and without C4-PI(3,4)P2 (black)for R43A (B) and K55A (C) are shown. Arg-43 is located outside and Lys-55 isinside the PI(3,4)P2-containing membrane-interacting interface. The fullspectrum of R43A is shown in supplemental Fig. S4B. D, fold increase in theassociation constant of the C4-PI(3,4)P2-p47phox PX interaction for each of thetested mutants, relative to that of the wild type. Association constant valueswere obtained from NMR titration curves.

TABLE 2Values of dissociation constants for the interaction of liposome-em-bedded C16-PI(3,4)P2 with WT and mutants of p47phox PX, obtainedfrom the CPM-based fluorescence assayThe numbers in parentheses denote the position of the CPM probe. CPM sites areindicated with arrows in Fig. 5A. Two variants with different positions ofCPMwere prepared to show that the presence of the probe does not affectthe binding affinity. The slightly higher dissociation constant for CPM-(65), as compared with that for CPM-(21), is in accordance with the bind-ing site being closer to site 65 than site 21. ND indicates not detectable.

CPM-labeledprotein Kd �2

�M

CPM-(21) 25 � 3 7.2 � 10�4

CPM-(21)T45A 38 � 6 22.9 � 10�4

CPM-(21)W80A 71 � 11 22.3 � 10�4

CPM-(21)K55E NDCPM-(21)R90L 42 � 6 25.6 � 10�4

CPM-(65) 86 � 30 14.6 � 10�4

CPM-(65)T45A 66 � 21 42.1 � 10�4

CPM-(65)K55A ND

FIGURE 6. Role for Lys-55 of p47phox in phagocyte oxidase activation.K562-gp91phox/p67phox cells were transfected with the cDNA for HA-taggedp47phox (WT) or p47phox (K55A). A, transfected cells (1.0 � 106 cells/ml) werepreincubated for 5 min at 37 °C and stimulated with phorbol 12-myristate13-acetate (PMA) (200 ng/ml). Chemiluminescence change by superoxideproduced was continuously monitored with DIOGENED, as described under“Experimental Procedures.” The superoxide scavenger superoxide dismutase(SOD) (50 �g/ml) was added at the indicated time. B, each graph representssuperoxide production estimated by chemiluminescence change (the aver-age S.D.) from four independent transfections. Protein levels of HA-p47phox,p67phox, and p22phox were analyzed by immunoblot with the anti-HA, anti-p67phox, and anti-p22phox antibodies, respectively, as described under “Exper-imental Procedures.”




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C4-PI3P, Arg-60 and Lys-92 of p40phox PX interact with thephosphates at position 3, 1, and 1 of C4-PI3P, respectively, andthese residues are not conserved in p47phox PX. Therefore, wefurther mutated the K55E p47phox PX at residues Thr-45(T45R) and Pro-78 (P78K). The T45Rmutation aims to restorethe role ofArg-60 in p40phoxPX, and the P78Kmutation aims torestore that of Lys-92 in p40phox PX (Fig. 7). The ligand-bindingsite and ligand specificity of the mutant were examined byNMR titration experiments with soluble C4-PI(3,4)P2 andC8-PI3P. As shown in Fig. 8, the resonances from a site thatcorresponds to the PI3P-binding site of p40phox PX wereaffected by the addition of C8-PI3P, whereas those correspond-ing to the phosphoinositide-binding site of the wild typep47phoxPX, determined by theTCS experiments (Fig. 2D), werenot affected. These results suggest that the T45R and P78Kmutations restored the PI3P-binding site of p40phox PX onp47phox PX. Importantly, most of these resonances were alsoaffected by the addition of C4-PI(3,4)P2 (Fig. 9 and supplemen-tal Fig. S9), suggesting that the mutation could not restore theabsolute PI3P specificity of p40phox PX.

DISCUSSION

NMR spectra provide structural information about proteinsat an atomic resolution. The position of the NMR signal, chem-ical shift, is directly affected by themicroenvironment. InNMRtitration experiments with soluble ligands, the binding of aligand causes the change of the microenvironment in the bind-ing site. In addition, the ligand binding can induce conforma-tional changes in the area located away from the binding site.Therefore, the chemical shift perturbation of the protein sig-nals is useful for the detection of the binding site for the ligandsand the site with the induced conformation change. The inten-sity reduction of the signal in the TCS experiment here indi-cates the membrane-interacting interface of the protein. Basedon the results of the TCS experiment, the C16-PI(3,4)P2-con-taining membrane-interacting interface on the p47phox PXdomain includes the residues from helix �1, helix �1�, and thefollowing loop up to the distorted left-handed PPII helix (Fig. 2,C and D). The mutational analyses verified the results of theTCS experiments and revealed that Lys-55 is indispensable forthe phosphoinositide recognition (Fig. 5). Lys-55 is located

inside the herein identified membrane-interacting interfaceand directly contacts the sulfate molecule in the crystal struc-ture of p47phox PX.

FIGURE 7. T45R/P78K/K55E mutant of p47phox PX. Solution structure of freep47phox PX (left) and crystal structure of p40phox PX with bound PI3P (right) areshown. In the structure of p47phox PX, only one of the two bound sulfates isshown. The Pro-78 and Thr-45 residues are shown in stick representations.Thr-45 and Pro-78 of p47phox PX correspond to Arg-60 and Lys-92 of p40phox

PX. They were mutated to Lys-78 and Arg-45, respectively, in an attempt torestore the Lys-92 and Arg-58 of p40phox PX.

FIGURE 8. Interaction of the T45R/P78K/K55E mutant of p47phox PX withC8-PI3P. A, HSQC spectrum of T45R/P78K/K55E mutant of p47phox PX before(black) and after (red) the addition of C8-PI3P. Representative residues fromthe typical and atypical binding site are colored green and blue, respectively.B, chemical shift perturbation profile of the T45R/P78K/K55E mutant ofp47phox PX caused by C8-PI3P. Normalized chemical shift changes and theirerrors were calculated with the same formula as in Fig. 3, but the color code isdifferent. Residues that showed strong (N �1.2 ppm) normalized chemicalshift changes are colored orange, and the broadened out residues are coloredred. Note that the scale of y axis is different. The chemical shift perturbationprofile is similar at critical points with a reported one for the Vam7p PX (49) inconformity with the presence of a typical binding site in both proteins.C, affected signals are mapped on the crystal structure of p47phox PX. Signalsbroadened beyond detection and broadened and shifted signals are coloredred and orange, respectively.




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The determined interface differs from those of all of the PXdomains elucidated so far, including the p40phox PX domain.Whereas the loss of the phosphoinositide binding activity ofp47phox PX by the K55E and R70Kmutations (Fig. 5 and Tables1 and 2) suggests that Lys-55 andArg-70 are the key residues forphosphoinositide binding, these residues are not conserved inthe other PX domains. Therefore, Lys-55 and Arg-70 would beresponsible for the unique phosphoinositide-binding site ofp47phox PX.The chemical shift perturbation area on p47phox PX, caused

by the addition of C4-PI(3,4)P2, is larger than the phospho-

inositide-binding site determined by the TCS experiments andincludes residues in the site that corresponds to the PI3P-bind-ing site of p40phox PX (Fig. 3, A and B). Considering that chem-ical shift perturbation is induced by both the direct bindingeffects and the conformational change (45), the perturbation ofthe residues in the site that corresponds to the PI3P-bindingsite of p40phox PX would be a secondary effect, due to a confor-mational change. The absence of the perturbation on the seg-ment of the � sheet comprising the C terminus of �1 and the Nterminus of the�2 strands, which also corresponds to the PI3P-binding site of p40phox PX and was perturbed in the titrationexperiments on p40phox and Vam7p (supplemental Fig. S5),supports the conclusion. The phosphoinositide-binding siteand the site that corresponds to the PI3P-binding site of p40phoxPX are adjacent, when projected not only on the tertiary struc-ture but also on the secondary structure of p47phox PX, becausethey are partly formed by the same structural features, includ-ing the �1 helix and the long loop that connects helices �1 and�2. Therefore, a conformational change would easily propagatefrom one site to the other.However, in the T45R/P78K/K55Emutant of p47phox PX, the

residues that correspond to the phosphoinositide-binding siteon p47phox PX were not perturbed, and the segment of the �sheet composed of the C terminus of �1 and the N terminus ofthe �2 strands was strongly perturbed (Figs. 8 and 9 and sup-plemental Fig. S9). Therefore, the T45R/P78K/K55E mutant ofp47phox PX would assume the p40phox-like phosphoinositide-binding site, whereas the phosphoinositide-binding site of thewild type p47phox PX differs from that of p40phox PX.

These results suggest that p47phox PX is unable to accommo-date a phosphoinositide in the area corresponding to that inp40phox PX, because the critical residues, such as Arg-60 andLys-92 of p40phox PX, which correspond to Thr-45 and Pro-78of p47phox PX, are absent (Fig. 7). We should point out that theP78Kmutation not only restored the essential role of Lys-92 inp40phox PX but also removed the residue Pro-78 that occupiesvaluable space inside the ligand binding pocket. This mightcontribute considerably to restoring binding capability (Figs. 8and 9 and supplemental Fig. S9). It could also explain why theadjacent residue Lys-79, which in the solution structure ofp47phoxPXpoints away from the binding pocket (Fig. 7), cannotassume the role of Lys-92 in p40phox PX. Therefore, such smallbut critical variations inside and outside the typical phospho-inositide binding pocket may cause the differences in theligand-binding sites of these two PX domains.Interestingly, our results can provide a rationale for the in

vivo behaviors of twomutants, those of Pro-73 andArg-70, thatare within the presently identified C16-PI(3,4)P2-containingmembrane-interacting interface. It has been reported that themutation Pro-73 abrogates phosphoinositide recognition andinterferes with the in vitro activity of NADPH oxidase (46),while the mutation Arg-70 prevents membrane translocationin HEK293 cells (47).Our experiments revealed that PA and soluble C4-PI(3,4)P2

competitively bind to the p47phox domain (Fig. 4). Therefore,the atypical ligand-binding site on p47phox recognizes both PAand PI(3,4)P2. This low specificity is in stark contrast with thestringent PI3P specificity of the typical binding site on p40phox.

FIGURE 9. Interaction of T45R/P78K/K55E mutant of p47phox PX withC4-PI(3,4)P2. A, excerpts of superimposed HSQC spectra of T45R/P78K/K55Emutant of p47phox PX before (black) and after (red) the addition of C4-PI(3,4)P2.Residues from the typical and atypical binding sites are colored green andblue, respectively. Full scale spectrum is shown in supplemental Fig. S9.B, chemical shift perturbation profile of the T45R/P78K/K55E mutant ofp47phox PX caused by C4-PI(3,4)P2. Normalized chemical shift changes andtheir errors were calculated with the same formula as in Fig. 3, but the colorcode is different. Residues that showed strong (N �2.9 ppm) normalizedchemical shift changes are colored orange, and the broadened out residuesare colored red. Note that the scale of the y axis is different. The chemical shiftperturbation profile is similar with a reported one for the Vam7p PX (49) inconformity with the presence of a typical binding site in both proteins. C, per-turbed signals are mapped on the crystal structure of WT p47phox PX. Thecoloring is the same as in Fig. 8B.




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Although at this point the physiological meaning of an atyp-ical phosphoinositide-binding site on the p47phox PX domain isunclear, the role of this atypical binding site should be impor-tant for the function of phagocyte NADPH oxidase becausemutation of Lys-55, a key residue of this binding site, signifi-cantly impedes normal production of superoxides (Fig. 6). Inthe same biological system, the phagocyte NADPHoxidase, thetwo PX domains, p47phox and p40phox PXs, differ in the loca-tions of the binding sites and in the affinities and selectivitiestoward phosphoinositides. These different structural proper-ties may hint at distinct functional roles. It has been shown thatin the assembled active NADPH oxidase complex, p40phox PXremains continually bound onmembrane-embedded PI3P dur-ing reactive oxygen species production (48). We speculate thatp47phox PX is not. As the phagosome matures and PI(3,4)P2gives space to PI3P, p47phox PXmay be released from themem-brane and reorient itself, relative to the other components ofthe active enzymatic complex. In this context, the shallownessof the atypical binding site, with its few binding determinants,may be a key structural feature that enables p47phox PX tointeract with multiple phosphoinositide signals, dependingon the stage of phagocyte NADPH oxidase assembly and/oractivation.It is worth mentioning that residues corresponding to His-

51, Lys-55, and Arg-70 (that contribute to the formation of theatypical binding site) and to Thr-45 and Pro-78 that contributeto the collapse of the typical binding site of human p47phox PXdomain are highly conserved among different species. They areidentical among organisms like human, bovine, pig, dog, andmouse. Thus, the atypical phosphoinositide-binding site doesnot appear to be an exclusive characteristic of the humanp47phox PX domain.Based on the dodecyl-phosphocholine titration experiments

of the Vam7 PX domain, Cheever et al. (49) proposed that thelong loop between helices �1 and �2 functions as a membraneinsertion loop. Importantly, the determined binding interfaceof p47phox PX does not include the speculatedmembrane inser-tion loop in the 77–86-residue region (14). Because the mem-brane insertion loop can affect the binding affinity, its existencewas examined further. It has been shown that the fluorescenceof IAEDANS, covalently attached tomembrane insertion loops,increases upon membrane insertion (50). The residues at posi-tions 21 and 80, which are located outside and inside the spec-ulatedmembrane insertion loop region, respectively, were sub-stituted with cysteines and labeled with IAEDANS. Both of theIAEDANS-labeled p47phox PX domains showed similar andonly weak fluorescence increases upon the addition of thePI(3,4)P2-containing liposomes, and themagnitude of the fluo-rescence increase was similar to that induced by control lipo-somes without PI(3,4)P2 (supplemental Fig. S10). In addition,the dodecyl-phosphocholine titration experiments with andwithout soluble C4-PI(3,4)P2 did not reveal an increased pro-pensity of the above region formembrane interactions (data notshown). Based on these results, we conclude that the 77–86-residue region of p47phox PX is not a membrane insertion loop.The 77–86-residue region of p47phox PX is much shorter andprotrudes less than themembrane insertion loop on theVam7pPX domain (Protein Data Bank code 1KMD). This area of

p47phoxPXmay nonspecifically contact with the lipid bilayer, asthe protein tries to accommodate the polar head of its ligand,but it does not function as a specific enhancer of membranebinding. Herein, the Kd value for the interaction of the p47phoxPX domain with liposome-embedded PI(3,4)P2 was estimatedto be 24 �M (fluorescence assay), which is comparable with theKd of 38.7�M (Table 1) for the soluble C4-PI(3,4)P2 (NMR titra-tion assay). The similarity of the above values is in agreementwith the absence of a membrane insertion loop that couldenhance the membrane docking. Glu-49 and Tyr-48, whichwere affected by the irradiation in the present TCS experi-ments, are spatially separated from the other affected residues(Fig. 2D). These residues might be important for a productiveinteraction with the lipid bilayer.The weak affinity of p47phox PX for membrane-embedded

PI(3,4)P2 is not necessarily incompatible with accurate mem-brane targeting. The combination of phosphoinositide recogni-tion by the PX domain and protein-protein interactions involv-ing other domains (such as the interaction of the two tandemSH3 domains with the proline-rich area of the integral mem-brane protein p22phox) can provide for accurate membrane tar-geting of the full-length p47phox. Such cooperation betweenphosphoinositide recognition and protein-protein interactionsfor membrane targeting has already been demonstrated forother proteins, including Vam7p (51). In the case of p47phox, itappears that protein-protein interactions are the primarymechanism of membrane targeting, because PX-truncatedp47phox is still able to find its way to the phagosomalmembrane(10). However, membrane targeting of isolated p47phox PXdomain in intact cells has not been reported and based on ourunpublished observations,3 isolated p47phox PX domain invari-ably fails in membrane localization. This conforms to its hereindemonstrated low affinity for phosphoinositides.The different patterns of phosphoinositide recognition

between p47phox PX and p40phox PX, despite their similar over-all structures, underscore how the substitution of the key resi-dues on the surface of the PX domain can significantly alter itsfunction. The functional alterations can be dramatic. The caseof the PX domains from phospholipase D1 and D2, which wereidentified as a GAP protein and an interacting partner forDynamin (52), is one of the first reports that challenged theconventional role of PX as merely a membrane interactingdomain.In conclusion, we performedTCS experiments that led to the

correct identification of the interacting interface ofmembrane-embedded PI(3,4)P2 on the p47phox PX domain. In similarapplications, the TCS experiments could provide insights intothe phosphoinositide recognition modes of various phospho-inositide-effector proteins, such as the PX, pleckstrin homol-ogy, ENTH, and C2 domains.

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Miyano, Hideki Sumimoto and Ichio ShimadaPavlos Stampoulis, Takumi Ueda, Masahiko Matsumoto, Hiroaki Terasawa, Kei

Phox Homology (PX) Domain Revealed by NMRphox)-binding Site on p472Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P

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