p47phox molecular activation for assembly of the neutrophil NADPH oxidase complex.

Abstract

The p47(phox) cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47(phox) activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22(phox) upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22(phox), reflecting an intermediate conformation between the autoinhibited and activated forms.

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p47phoxMolecular Activation for Assembly of the Neutrophil
NADPH Oxidase Complex*□
Receivedforpublication,April30,2010,andinrevisedform,June28,2010 Published,JBCPapersinPress,June30,2010,DOI10.1074/jbc.M110.139824
Julien Marcoux‡§¶?, Petr Man§¶?**‡‡, Isabelle Petit-Haertlein‡¶?, Corinne Vive `s‡¶?, Eric Forest§¶?, and Franck Fieschi‡¶?1
Fromthe‡LaboratoiredesProte ´inesMembranairesand§LaboratoiredeSpectrome ´triedeMassedesProte ´ines,Commissariata `
l’EnergieAtomique(CEA),DirectiondesSciencesduVivant,InstitutdeBiologieStructurale(IBS),41rueJulesHorowitz,Grenoble,
F-38027,France,¶CNRS,UMR5075,F-38027Grenoble,France,the?Universite ´ JosephFourier,F-38041Grenoble,France,the
**InstituteofMicrobiology,AcademyofSciencesoftheCzechRepublic,v.v.i.,Videnska1083,CZ-14220Prague4,CzechRepublic,
andthe‡‡DepartmentofBiochemistry,FacultyofScience,CharlesUniversityinPrague,Hlavova8,CZ-12840Prague2,CzechRepublic
S
The p47phoxcytosolic factor from neutrophilic NADPH oxi-
dasehasalwaysbeenresistanttocrystallogenesistrialsduetoits
modular organization leading to relative flexibility. Hydrogen/
deuterium exchange coupled to mass spectrometry was used to
obtain structural information on the conformational mecha-
nism that underlies p47phoxactivation. We confirmed a relative
opening of the protein with exposure of the SH3 Src loops that
are known to bind p22phoxupon activation. A new surface was
showntobeunmaskedafteractivation,representingapotential
autoinhibitory surface that may block the interaction of the PX
domainwiththemembraneintherestingstate.Withinthissur-
face,weidentified2residuesinvolvedintheinteractionwiththe
PXdomain.ThedoublemutantR162A/D166Ashowedahigher
affinity for specific phospholipids but none for the C-terminal
part of p22phox, reflecting an intermediate conformation
between the autoinhibited and activated forms.
NADPH oxidases (Nox) and Dual oxidases (Duox) are mul-
tienzymatic complexes found in many cell types (1) that play a
wide range of physiological roles (2). The seven different iso-
forms of these enzymatic complexes differ in their membrane
redox components: Nox1 to Nox5, Duox1 and Duox2. Despite
this molecular heterogeneity, they all share the common fea-
tureofreactiveoxygenspeciesproduction,whichiseithercon-
stitutive (Nox4) or inducible by cytosolic factors (Nox1, Nox2,
and Nox3) or Ca2?(Nox5, Duox1, and Duox2).
The neutrophilic isoform containing Nox2, formerly called
gp91phox, is often referred to as a specialized reactive oxygen
speciesproducerasitsmainroleistotriggertheoxidativeburst
in neutrophilic phagosomes leading to the killing of phagocy-
tizedpathogens.Ofcourse,thiscatalyticactivitymustbetightly
regulated to avoid excessive reactive oxygen species produc-
tion, leading to oxidation of macromolecules, DNA mutations,
aging, and cell death. In contrast, a defect in NADPH oxidase
activity results in chronic granulomatous disease, an inherited
immunodeficiencycharacterizedbyanabnormalinflammatory
responseandrecurrentbacterialandfungalinfections(3).This
tight regulation is enabled by a physical separation before acti-
vation between the membrane-related flavocytochrome b558
(heterodimer of Nox2 and p22phox), a small G protein (Rac1 or
Rac2),andthecytosolicfactorsp40phox,p47phox,andp67phox.In
the resting state, these modular proteins form a heterotrimeric
complex based on different interactions between their con-
served binding domains (4); p40phoxand p67phoxinteract via
their respective PB1 domains (5, 6), and the p47phoxproline-
rich region binds the p67phoxC-terminal SH32domain (7).
The p47phoxsubunit is a key component in the activation
process. It contains an N-terminal phox homology domain
(PX) and SH3 domains arranged in tandem and ends with a
proline-rich region (see Fig. 1). In the whole NADPH oxidase
assembly, it acts as a sensor of the activation signal through
multiple phosphorylations on serines within its autoinhibitory
region (AIR). The activated form of p47phoxdrives the trans-
location of the other cytosolic factors for assembly onto fla-
vocytochrome b558through the following mechanisms. Upon
activation, the PX and tandem SH3s domains promote assem-
bly through interactions with phosphoinositides (8–12) and
p22phoxC-terminal proline-rich region (13–15), respectively.
To avoid any constitutive binding to the membrane and main-
tain the cytosolic location of p47phox, the tandem SH3s and PX
domains must be masked in the resting state. Two functional
states of p47phoxhad already been proposed, corresponding to
both activated and autoinhibited conformations.
The first p47phoxautoinhibited model suggested an interac-
tion between the tandem SH3s and some downstream residues
(13),latelydefinedastheAIR.Sincetheoriginalproposal,many
studies have tried to decipher the relative organization of the
different domains in the autoinhibited state. Concomitantly
withthisfirstinteractionarosethehypothesisofanotherinter-
action between the PX and SH3B domains (9), which was
widelyacceptedinthefield(4,7,8,16,17).Newinsightsintothe
molecularmechanismofinhibitionwereprovidedbythestruc-
tureofthetandemSH3sinteractingeitherwiththeAIRorwith
* This work was supported by grants from the CEA and the Association pour
la Recherche sur le Cancer.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Table S1 and Figs. S1–S4.
1To whom correspondence should be addressed: IBS, 41 rue Jules Horowitz,
Grenoble F-38027, France. Tel.: 33-4-3878-91-77; Fax: 33-4-38-78-54-94;
E-mail: franck.fieschi@ibs.fr.
2Theabbreviationsusedare:SH3,Srchomology3;phox,phagocyteoxidase;
AIR, autoinhibitory region; PX, Phox homology; SPR, surface plasmon
resonance; DXMS, deuterium exchange coupled to mass spectrometry;
PtdIns(3,4)P2, phosphoinositol-3,4-biphosphate; POPC, 1-palmitoyl-2-
oleyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleyl-sn-glycero-
3-phosphoethanolamine; POPA, 1-palmitoyl-2-oleyl-sn-glycero-3-phos-
phate; Cter, C terminus; TM, transmembrane.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 37, pp. 28980–28990, September 10, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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the C-terminal part of p22phox(18). However, this result was
totally incompatible with the interaction between the PX and
SH3B domains because both interactions involve the same
binding region in the tandem SH3s, as pointed out by several
groups (17–20).
Recently, our group proposed a model for the global confor-
mation of the entire p47phoxin the autoinhibited form, using
small angle x-ray scattering (20). It allowed us to definitely dis-
cardthehypothesisofaninteractionbetweenthePXandSH3B
domains. p47phoxsmall angle x-ray scattering envelopes sug-
gested that the PX domain was located in the proximity of the
firstSH3domain(SH3A),butitwasnotpossibletoconcludeon
whether the PX interacts directly with the SH3A or is main-
tained in close proximity by a structured linker. Moreover, this
work has strengthened the AIR-tandem SH3s interaction
hypothesis initially based on studies using p47phoxisolated
modules. The AIR release from the tandem SH3s, occurring
upon activation, has recently been confirmed by limited prote-
olysis coupled to mass spectrometry (21). Coupling between
the AIR release and enhanced lipid binding affinity was shown,
suggesting changes in PX domain accessibility. Deuterium
exchangecoupledtomassspectrometry(DXMS)performedon
the entire proteins confirmed the conformational difference
betweentheactivatedandautoinhibitedstate(21).However,
conclusive data regarding the molecular mechanism of PX
inhibitionintherestingstateandmorepreciselyproofsofits
involvement in any intramolecular interaction were still
missing.
Togetdetailedinsightintothestructuralchangesofp47phox,
we extended our previous study and followed local deuteration
kineticsintheindividualpartsoftheproteininbothstates.We
confirmedandmorepreciselydescribedtheexposureoftheSrc
loops, from both SH3s, due to AIR release. Remarkably, for the
first time, we identify a novel surface within the SH3A domain
that is unmasked during activation. Using site-directed
mutagenesis and the liposome binding assay, we confirm that
this region is involved in the inhibition of the PX domain lipid
binding properties.
This study contributes a final response to the question of
the p47phoxautoinhibitory mechanism. The modular organi-
zation deciphered here, involving up to four regions, has
never been reported, particularly regarding PX and SH3
domain interaction.
EXPERIMENTAL PROCEDURES
Materials—Glutathione-Sepharose high performance and
SP Sepharose high performance columns were from GE
Healthcare (Little Chalfont, Buckinghamshire, UK). Jupiter
C18 column (50 ? 1 mm; 5 ?m, 300 Å) was from Phenomenex
(Torrance, CA). C4 and C8 macro traps were from Michrom
Bioresources, Inc. (Auburn, CA). Pepsin from porcine gastric
mucosa and protease type XIII from Aspergillus saitoi were
from Sigma-Aldrich. The following products were purchased
from Avanti Polar Lipids (Alabaster, AL): 1-palmitoyl-2-oleyl-
sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleyl-sn-
glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleyl-
sn-glycero-3-phosphate (POPA), and 1,2-dioleoyl-sn-glycero-
3-(phosphoinositol-3,4-biphosphate) (PtdIns(3,4)P2).
ProteinCloning,Expression,andPurification—cDNAencod-
ing p47phoxresidues 1–390 (p47phox) or 1–342 (p47phox?Cter)
and the cytosolic region of p22phox(residues 132–195) were
clonedintopGex-6Pvectors,addinganN-terminalGSTfusion
tag (see Fig. 1). Mutations were introduced by PCR-mediated
site-directed mutagenesis. Due to the genetic construct, the
full-lengthp47phoxharbors7additionalresiduesattheCtermi-
nus. All constructs were sequenced to confirm their identities.
p47phox, p47phox?Cter, and GST-p22phoxCter constructs
were expressed in Escherichia coli BL21(DE3) and purified
according to Durand et al. (20), with slight differences de-
scribed hereafter. Lysis buffer for p47phoxpurification included
1 mM EDTA, but we used 2 mM MgCl2supplemented with
DNase and Complete EDTA-free protease inhibitor (Roche
Diagnostics, Basel, Switzerland) for GST-p22phoxCter purifica-
tion. Buffers were set at pH 7.5 for p47phoxconstruct purifica-
tion and at pH 7 (lysis buffer) and pH 8 (elution buffer) for
GST-p22phoxCter purification. GST cleavage gave rise to
p47phoxencoding residues 1–342, which will be referred to as
p47phox?Cter.GST-p22phoxCterwasnotcleavedafterglutathi-
one-Sepharose elution but was reloaded twice on the same
column to bind any residual GST and concentrated on an
Amicon centrifugal device with a 10-kDa cutoff (Millipore,
Billerica, MA).
GST used for surface plasmon resonance (SPR) experiments
was obtained after GST-p47phox?Cter overnight cleavage with
PreScissionprotease(GEHealthcare).ItwasrecoveredfromSP
Sepharose column flow-through and concentrated on an Ami-
con centrifugal device with a 10-kDa cutoff.
p47phoxtandem SH3s was also purified according to Durand
et al. (20), with slight differences. The culture pellet was resus-
pended in 50 mM Tris, pH 7, 300 mM NaCl, 2 mM EDTA, 4 mM
1–4-dithiothreitol(DTT)inthepresenceofCompleteprotease
inhibitor. After overnight digestion at 4 °C with PreScission
protease,thecleavedproteinwasprecipitatedwith60%ammo-
nium sulfate and purified using a Superdex 75 prep grade col-
umn (GE Healthcare). The p47phoxtandem SH3s was finally
eluted as a dimer, and the fractions were pooled and concen-
trated to 2.5 mg/ml on an Amicon centrifugal device with a
10-kDa cutoff.
Surface Plasmon Resonance—SPR experiments were con-
ducted using a Biacore 3000 instrument (Biacore AB, Paris,
France) equipped with a CM4 sensor chip. The machine was
primed with the running buffer (137 mM NaCl, 2.7 mM KCl, 8.1
mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4, and 0.005% surfactant
P20). p47phoxwas diluted in the running buffer to the required
concentrations reported in Fig. 2. Before immobilization, 70 ?l
of N-hydroxysuccinimide and 70 ?l of 1-ethyl-3-(3-dimethyl-
aminopropyl)-carbodiimide were first mixed, and 50 ?l of the
resulting mix were injected to prime the surface at 5 ?l/min, as
recommendedbythemanufacturer.Tenmicrolitersof78.2nM
GST-p22phoxCter diluted in 4 mM sodium acetate, pH 5, were
then immobilized at 5 ?l/min, and 30 ?l of ethanolamine were
finallyinjectedtosaturatethesurface.Allrunswerecarriedout
at20 °Candataflowrateof20?l/min.Aftereachinjection,the
surfacewasregeneratedbya10-?linjectionof10mMNaOHat
a flow rate of 20 ?l/min. For each p47phoxinjection (60 ?l), a
blank was run in parallel on a surface functionalized with 78.2
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nM GST to subtract nonspecific binding. For the p47phox?Cter
and p47phoxTM-?Cter comparison, we carried out crossed
scales to overcome the possible problems of surface degrada-
tion over time by alternating injections between p47phox?Cter,
p47phoxTM-?Cter, and p47phoxtandem SH3s. For point muta-
tion analysis, crossed scales between p47phoxtandem SH3s and
themutantsanalyzedwerecarriedout.Foreverythirdfull-scale
analysis,newCM4surfaceswerefunctionalizedwithbothGST
and GST-p22phoxCter.
Multilamellar Vesicle Binding Assay—The in vitro semi-
quantitative liposome binding assay was adapted from Ago et
al. (8) with minor modifications. Liposomes were prepared by
mixing POPC and POPE (50:50) for the control (MLV1) and
POPC, POPE, POPA, and PtdIns(3,4)P2(45:45:5:5) for specific
liposomes(MLV2).Themixturewasthendriedundernitrogen
and resuspended to a concentration of 2 mM of total lipids in a
binding buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM DTT).
After 2 h of incubation on ice, liposomes were obtained by vor-
texing. Proteins (5 ?M) were incubated with liposomes (1 mM)
for 15 min at 20 °C in 100 ?l of binding buffer. Liposomes were
collected by ultracentrifugation (1 h at 100,000 ? g in an Air-
fuge rotor). Aliquots of bound (liposome pellet) and unbound
p47phox(supernatant) were taken for further analysis by 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Coomassie Blue-stained gels were scanned and
analyzed by densitometry. Three independent experiments
were conducted.
H/D Exchange and Data Analysis—Deuteration of
p47phox?Cter and p47phoxTM-?Cter was initiated by a 20-fold
dilution into a deuterated buffer (5 mM Hepes, pD 7.4, 1 mM
EDTA, 2 mM DTT, and 200 mM NaCl). Aliquots (40 ?l) were
taken after 30 s, 1 min, 5 min, 10 min, 30 min, 1 h, 3 h, 5 h, 6 h,
and8hforthelocalkineticsand10s,30s,1min,5min,10min,
30 min, and 1 h for the global kinetics. The exchange was done
at 21 °C and was quenched by the addition of 5.6 ?l of 50 mM
HCl and rapid freezing in liquid nitrogen. Each sample was
quickly thawed and digested on ice for 2 min with pepsin
(enzyme/protein w/w ratio 1) or protease type XIII (enzyme/
protein w/w ratio 12) before mass spectrometric analysis.
HPLC Separation—The digestion mixture was loaded on a
peptide MacroTrap and desalted with 0.03% trifluoroacetic
acid (TFA: solvent A) for 1 min at a flow rate of 400 ?l/min.
After that, peptides were eluted with a linear gradient (from 17
to 45% solvent B in 20 min, where solvent B was 95% acetoni-
trile, 0.03% TFA).
Mass Spectrometric Analysis—Peptide sequencing was
performed using a quadrupole ion trap mass spectrometer
(ESQUIRE 3000?, Bruker Dalton-
ics) equipped with an electrospray
source. For the MS/MS experi-
ments, the three most intense ions
from the preceding MS scan were
fragmented. Tandem mass spectra
were searched using Mascot, and
theassignments
manually and by accurate mass
measurements.
Accurate mass
were verified
measurements
and the analysis of the local kinetics of deuteration were done
on a time-of-flight (TOF) mass spectrometer (6210, Agilent
Technologies, Santa Clara, CA) equipped with an electrospray
source. Data were processed with Analyst QS and MassHunter
Qualitative Analysis, and the deconvolution and calculation of
the average masses were carried out in Magtran (22). Deutera-
tion percentages (%D) were calculated using Equation 1
%D ? ??mx%? m?/N? ? 100
(Eq. 1)
wheremandmx%representtheaveragemolecularmassofnon-
deuterated and partially deuterated samples, respectively, and
N represents the number of exchangeable amide protons. Two
independent measurements were taken, and the data were
averaged. Data were processed with the scripts available at the
Laboratory of Molecular Structure Characterization (IMIC,
Prague) MS Tools web page.
RESULTS
p47phoxTM-?Cter Interacts with the C Terminus of p22phox—
To study the structural modifications occurring in p47phox
upon activation, we previously designed two recombinant
forms corresponding to the autoinhibited and activated states
(21). These p47phoxconstructs are C-terminally truncated but
still contain all the domains involved in the activation process
(PX domain, tandem SH3s, and AIR) (Fig. 1). This C-terminal
truncation(fromresidue343)improvesthehomogeneityofthe
purifiedproteinwithoutaffectingthestructuralintegrityofthe
protein, as published previously (23).
To mimic serine phosphorylations occurring upon p47phox
activation, the corresponding serines 303, 304, and 328 were
mutatedintoglutamicacid.Weandothershaveshownthatthis
triple mutant (p47phoxTM-?Cter) presented an enhanced
binding affinity for phosphoinositide lipids when compared
withp47phox?Cter(8,16,21).Inaddition,structuralanalysisby
deuterium exchange and limited proteolysis coupled to mass
spectrometry also showed that this p47phoxTM-?Cter pre-
sented a more open conformation, notably through release of
the AIR (21). These previous characterizations supported the
notion that p47phoxTM-?Cter is a good mimic of the activated
state.However,asaprerequisitetopursuingthedetailedstruc-
tural characterization of these two constructs, we needed to
fully validate that the p47phoxTM-?Cter is able to interact with
p22phox, as described earlier on the full-length p47phox(18, 24).
SPRwasusedtoquantifythisinteraction.CM4surfaceswere
functionalized with a GST-p22phoxCter construct (Fig. 1) or
GST alone (negative control). Increasing p47phoxtandem SH3
FIGURE 1. Molecular constructs used in this study. PRR, proline-rich region.
Decipheringthep47phoxActivationMechanism
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concentrations were used as positive control experiments to
compare with other p47phoxconstructs. No significant interac-
tion with the GST-p22phoxCter surface could be detected using
p47phox?Cter, confirming its autoinhibited state (Fig. 2). The
triple mutation S303E/S304E/S328E was sufficient to recover
an affinity similar to that of p47phoxtandem SH3s alone: 187 nM
versus69nM(supplementalFig.S1andsupplementalTableS1).
Similar results were obtained with the full-length forms of the
protein; no interaction between GST-p22phoxCter and p47phox
was observed, and the dissociation constant was determined at
55nMforthefull-lengthp47phoxTM(datanotshown).Thelack
ofaffinityofwild-typep47phoxconstructswhencomparedwith
the S303E/S304E/S328E triple mutants clearly shows the con-
formational change occurring during p47phoxactivation and
validates the use of p47phoxTM-?Cter as a true mimic of the
activated state.
Peptide Mapping—Proteolysis with acidic proteases was
usedtolocalizethedifferencesindeuterationbetweentheacti-
vated and autoinhibited forms of p47phox. Due to ragged and
nonspecific digestion, the generated peptides had to be identi-
fied by means of tandem mass spectrometry. The separate use
of pepsin and protease type XIII generated 89 cleavage sites,
providing a total of 127 peptides with only five occurring in
both digests (supplemental Fig. S2). The combination of pep-
tides generated by both proteases provided 99% sequence cov-
erage. Generated peptides have an average size of 14 residues.
Theaveragenumberofcleavagesitesandgeneratedpeptidesin
the AIR was twice as low (17 cleavage sites and 23 peptides per
100 residues) as in the rest of the protein where a rather good
resolution was obtained (28 cleavage sites and 40 peptides per
100residues).ThepolybasicnatureoftheAIRismostprobably
behind the low spatial resolution in this region. Although pep-
sinisknowntopreferhydrophobicsites(25)andthusprovided
onlylongpeptides,fungalproteaseXIIIwasshowntohavepref-
erence for basic amino acids (26) and thus most probably gen-
erated very short peptides that were not detected.
Solvent Accessibility of Different p47phox?Cter Regions—The
exchange kinetics of individual p47phox?Cter regions were fol-
lowed on 30 peptides from the pepsin digest and 10 from the
protease XIII digest, together covering 92% of the sequence.
The missing 7% are due to the 4 N-terminal residues and to the
gaps coming from the fact that we do not monitor exchange on
the N-terminal amine of the peptides. Peptides used for DXMS
studyareshowninsupplementalFig.S2,anddeuterationkinet-
ics for selected peptides are shown in supplemental Fig. S3.
Sevenregionsweredeterminedbythedifferencebetweenover-
lapping peptides. As an example, the deuteration percentage of
region276–279wasobtainedbysubtractingpeptides264–275
data from peptides 264–279 data (supplemental Fig. S4A).
Moreover, spatial resolution was increased by the presence of
short peptides. For instance, information given by peptides
161–174 was improved by the combined use of peptides 161–
166 and 167–174 (supplemental Fig. S4, B–D).
Thefirstinformationgivenbythelocalkineticsisthesolvent
accessibility of different p47phoxregions (supplemental Fig.
S4A). As expected, the C-terminal region of p47phox(residues
333–342) was quickly deuterated, as was the linker region
between the PX domain and the first SH3 (residues 119–160).
As shown by the protection plots (Fig. 3, A and B), these two
regions are already deuterated more than 50% after only 30 s of
deuterium exchange. This is in complete agreement with the
predictedunfoldednatureofthesesegments(20).Itisalsosup-
ported by our recent results obtained by limited proteolysis
(21). Even if it seems poorly compatible with the structural and
functional role that has recently been suggested for this linker
(23), the latter one cannot be excluded as other structured
regions show fast deuteration kinetics (e.g. helix 3 from PX;
supplemental Fig. S4A). Regarding the AIR, fast deuteration
kinetics are also observed, as expected from the structure, due
FIGURE 2. Surface plasmon resonance results obtained for p47phoxtandem
SH3s, p47phoxTM-?Cter, and p47phox?Cter on CM4 chips functionalized
with GST-p22phoxCter. Increasing concentrations (from 40 nM to 2.56 ?M) of
eachconstructwereinjected.R.U.,resonanceunits.
Decipheringthep47phoxActivationMechanism
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to its unfolded sequence exposed at the surface of the tandem
SH3s (18).
Half of the secondary structural elements described by the
crystallographic and NMR results (9, 16, 18, 19, 27), such as
sheets1andhelices1–4fromPXandsheetsB-C-DfromSH3A
and SH3B, show slow deuteration kinetics, as expected. The
other ?-helices and ?-sheets do not show such slow deuterium
incorporation, probably because corresponding segments are
covered by peptides including both unstructured and struc-
tured region (e.g. peptides 279–289).
FIGURE3.LocalkineticsofH/Dexchange.AandB,protectionplotsshowingthedeuterationlevelsofp47phox?Cter(blue)andp47phoxTM-?Cter(red)after30s
(A) and 8 h (B) of deuteration. PRR, proline-rich region. C, difference of deuteration between the two constructs after 8 h of deuteration: ?%D ? % of
p47phoxTM-?Cter ? % of p47phox?Cter. cyan, ? 8%; yellow, ?15%; orange, ?25%, red, ?35%.
Decipheringthep47phoxActivationMechanism
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Deuterium Exchange Comparison between Autoinhibited
andActivatedForms,IdentificationofUnmaskedSurfacesupon
Activation—Structural changes occurring upon p47phoxac-
tivation are best evidenced by examining the differences
between
p47phox?Cter deuteration percent-
ages(Fig.3C).Actually,upondeute-
rium incorporation, the isotopic
distributions of the peptides are
shiftedtothehighermasses,andfor
some peptides, such as 161–166 or
255–263 (Fig. 4), the incorporation
is faster for p47phoxTM-?Cter than
for p47phox?Cter. This faster deute-
rium incorporation for p47phoxTM-
?Cter can be directly interpreted as
better accessibility to the solvent
following conformational changes.
Five regions, all located within the
tandem SH3s, showed increased
deuteration rates for p47phoxTM-
?Cter, whereas no significant dif-
ferences were highlighted in the PX
domain. Deuteration differences
between both autoinhibited and
activated forms of p47phoxwere
plottedontotheavailablestructures
of the “tandem SH3s-AIR super-
complex” and of the PX domain
(Fig. 5A) (18). When looking at the
tandem SH3s, two different events
canbedistinguishedtointerpretthe
observed unmasked surfaces in the
activated form.
The first one corresponds to pep-
tides encompassing residues 186–
194 from SH3A and 254–263 from
SH3B. These two regions, facing
each other, are the two Src loops
that are known to be exposed to the
solvent after activation to bind the
p22phoxCterpart(13,14,24,28–31).
Two other regions within SH3B
(234–236 and 276–279) are pro-
tectedintheautoinhibitedstateand
formaninnersurfacemaskedbythe
AIR. In all these regions, the better
accessibility to the solvent upon
activation can be explained by the
release of the AIR, as illustrated in
Fig. 5, B and C.
Secondly, another surface ex-
posed upon p47phoxactivation was
detected by DXMS (residues 162–
166, 176–185, and 195–209). This
regionencompasses
SH3A lateral surface (Fig. 5, D and
E), which has never been suggested
p47phoxTM-?Cterand
thewhole
to be involved in the activation process. In a recent study of the
p47phoxresting state, crystallographic structures of the PX
domain and tandem SH3s (16, 18) have been fitted in the low
resolution envelopes of the whole protein obtained by small
253-263
708709710711712713714 m/z
541542543544545 546 m/z
,
Normalized ESI-MS Intensity
30 sec
30 min
6 hrs
161-166255-263
1e+11e+21e+31e+41e+5
% deuteration
20
40
60
80
1e+11e+2 1e+31e+41e+5
% deuteration
20
40
60
80
161-166
FIGURE 4. Examples of peptides with differential kinetics of H/D exchange depending on the p47phox
forms.A,detailsofisotopicprofilesoftwoselectedpepticpeptides(161–166and255–263)fromp47phox?Cter
(blue) and p47phoxTM-?Cter (red) demonstrating shifts toward higher masses during the deuteration. Three
timepoints,30s,30min,and6h,areshown.ESI-MS,electrospraymassionization-MS.B,deuterationkineticsof
thetwopeptidesshowninpanelAplottedasthepercentageofdeuterationversustimeforp47phox?Cter(blue
circles) and p47phoxTM-?Cter (red circles). Values are the average of two independent experiments.
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angle x-ray scattering (20). From the derived domain arrange-
ment,thesurfaceidentifiedhereispotentiallylocalizedinclose
vicinity of the PX domain in the autoinhibited state. The
unmasking of these regions coupled with the enhancement of
lipid binding properties strongly support our initial proposal
that the SH3A lateral surface is involved in the PX domain
locking in the resting state (Fig. 5A, arrow).
We expected to see a deuteration difference within the PX
domainandtheAIRbecausepreviousworkclearlyshowedthat
the PX domain and AIR release upon activation (21). The
absence of difference in the PX can be explained by differences
located for the most part at the pro-
tein surface that cannot be detected
byDXMSaslateralchainhydrogens
are not monitored.
R162A/D166A Mutations in the
SH3ALateralSurfaceInducethePX
Domain Release—To confirm the
directinteraction
SH3A and the PX domain through
the surface highlighted by DXMS, a
mutagenesis study was conducted.
Some of the residues exposed to the
solvent, potentially responsible for
this interaction (Fig. 5, A and E),
were chosen. The following single
and double mutations were made:
R162A,D166A,
Q197A,K199E,andR202A.Binding
to the C-terminal part of p22phox
was tested for each mutant using
SPR analysis. In contrary to the tan-
dem SH3s, none of these mutants
were able to interact with p22phox
(Fig. 6, A and B, and data not
shown). This proves that the tan-
demSH3s-AIRsupercomplexisnot
disrupted in mutants of this lateral
surface.
ReleaseofthePXdomainforeach
mutant was determined by pull-
down assay with liposomes, as
described previously (21). Of the
tested mutants, only R162A/D166A
showed drastically enhanced bind-
ing properties with specific phos-
pholipidsPtdIns(3,4)P2
compared with p47phox?Cter (Fig.
6C). The structural integrity of
p47phox?Cter,
and
was confirmed by circular dichro-
ism (Fig. 6D). These data suggest a
specificroleofresiduesArg-162and
Asp-166 in PX domain inhibition.
Tohighlighttheimportanceofboth
residues in the maintenance of the
p47phoxautoinhibited state, the glo-
betweenthe
R162A/D166A,
when
p47phoxTM-?Cter,
p47phoxR162A/D166A-?Cter
bal kinetics of deuteration were determined, as described pre-
viously(21).Thep47phoxR162A/D166A-?Cterdeuterationrate
is in between the rates of p47phox?Cter and p47phoxTM-?Cter
(Fig. 6E). This result reflects an intermediate conformation of
this mutant that has never been described. Indeed, it possesses
a closed tandem SH3s-AIR supercomplex with an open PX
domain accessible for lipid binding.
DISCUSSION
The molecular activation of the NOX2-based NADPH oxi-
dase has been the subject of intense studies over the last few
FIGURE5.LocalkineticsofH/Dexchangetransferredontothecrystallographicstructuresoftheautoinhib-
itedSH3tandem(PDB:1NG2)(18)andPXdomain(PDB:1O7K)(16).Differenceofdeuterationbetweenthetwo
constructsafter8hofdeuteration:?%D?%ofp47phoxTM-?Cter?%ofp47phox?Cter.cyan,?8%;yellow,?15%;
orange, ?25%; red, ? 35%. A, PX domain and tandem SH3s-AIR supercomplex structures correspond to residues
1–123and157–332ofp47phox,respectively.Undeterminedstructuresofflexibleregionsarerepresentedasdashed
lines, and mutated residues are represented as sticks. The sulfates bound in the phospholipid-binding sites are
showninstickrepresentation(green).BandC,thesurfaceoftheautoinhibitedtandemSH3sisrepresentedwith(B)or
without(C)theAIR,mimickingtherestingandactivatedstates,respectively.DandE,lateralviewoftheautoinhib-
itedtandemSH3sshowingthehighlightedregionasasurface(D)orassticks(E).
Decipheringthep47phoxActivationMechanism
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decades. Considering the level of reactive oxygen species
potentially produced by this specialized system, tight regula-
tion is of primary importance. Indeed, an abnormal increase in
this activity can lead to dramatic consequences, as observed in
neurodegenerative pathologies (such as Alzheimer disease,
Parkinson disease, HIV dementia, and demyelinating dis-
ease) (2). Therefore, better knowledge of the molecular
mechanism behind this tightly regulated system is highly
important, not only for basic knowledge but also to advance
toward therapeutic strategies for better control of these
pathological deregulations.
Two main events are central in the activation of NADPH
oxidase. Flavocytochrome b558requires direct interaction with
Rac1/2 and p67phoxto trigger the
electron transfer (32, 33). The pres-
ence of p67phoxat the membrane
requires conformational changes of
p47phoxthat will orchestrate their
co-translocation from the cytosol.
Despite the crucial role played by
these two activation steps, the
related molecular mechanisms are
poorly understood.
brings new insights into the struc-
tural changes undergone by p47phox
upon activation.
Interaction of Activated p47phox
with p22phox—It was shown that
p47phoxTM-?Cter
properties are enhanced in contrast
to p47phox?Cter (21). However, its
capacity to interact with p22phox
remained to be confirmed. The
interactionpropertiesoftheautoin-
hibited (p47phox?Cter) and acti-
vated state mimic (p47phoxTM-
?Cter) with GST-p22phoxCter have
thus been addressed by SPR. As
expected, p47phox?Cter did not
interact,whereasp47phoxTM-?Cter
showed interaction properties that
were similar to the p47phoxtandem
SH3s (positive control, Fig. 2). The
interactionbetweenp47phoxandthe
C-terminal region of p22phoxhas
already been effectively described
fromthebiochemical(13,18,24,28,
30,31)andstructural(14,29)points
of view.
Concerning tandem SH3s, the
dissociationconstantrelativetothis
interaction using isothermal titra-
tion calorimetry (18) and fluores-
cence titration methods (18, 29) led
to Kdvalues of 0.19, 0.40, and 0.64
?M, respectively. As for full-length
p47phoxTM, a higher Kdvalue was
determined by fluorescence titra-
Ourstudy
lipidbinding
tion at 17.8 ?M (18). These affinities are lower than the affinity
reported here, with a Kdestimated at 69 nM for the tandem
SH3s and 187 nM for the p47phoxTM-?Cter. This difference
might be attributed to the use of a p22phoxC terminus, which is
three times longer (63 residues versus 20) than reported in pre-
vious studies. The C terminus of p22phoxused in this study
corresponds to the entire cytosolic region following the last
putativetransmembranesegment.Theimprovementinaffinity
suggests that the additional residues, apart from the canonical
proline-rich region, may also play a role in stabilizing the
p47phox/p22phoxinteraction. However, the affinity obtained
with different analytical methods, p47phoxconstructs, and
p22phoxCter peptides of various lengths may only be compared
FIGURE6.R161A/D166AmutationsinduceselectiveopeningofthePXdomain.AandB,surfaceplasmon
resonance results obtained for p47phoxtandem SH3s (A) and p47phoxR162A/D166A-?Cter (B) on CM4 chips
functionalizedwithGST-p22phoxCter.Increasingconcentrations(from40nMto2.56?M)ofeachconstructwere
injected. R.U., resonance units. C, phosphoinositide binding activity of p47phox?Cter, p47phoxTM-?Cter,
p47phoxK199E-?Cter, p47phoxQ197A-?Cter, p47phoxR202A-?Cter, p47phoxR162A/D166A-?Cter, p47phoxR162A-
?Cter, and p47phoxD166A-?Cter. All proteins were incubated with liposomes containing POPC:POPE 50:50
(MLV1)orPOPC:POPE:POPA:PtdIns(3,4)P245:45:5:5(MLV2).SandPareliposomalsupernatantandpelletafter
centrifugation, corresponding to the unbound and bound fraction, respectively. Samples were analyzed by
SDS-PAGE and quantified by densitometry. Error bars indicate S.E. D, circular dichroism of p47phox?Cter,
p47phoxR162A/D166A-?Cter, and p47phoxS303/304/328E-?Cter. E, deuteration kinetics for p47phox?Cter (black
triangles,grayline),p47phoxTM-?Cter(blacksquares),andp47phoxR162A/D166A-?Cter(opencircles,dashedline)
at pD 7.4.
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withgreatcaution.Themostimportantinformationhereisthe
ability to discriminate between open and closed tandem SH3s,
reflecting the activated or the autoinhibited states of the
protein.
Deciphering the Molecular Opening of p47phox—The DXMS
localkineticspresentedhereareanaturalfollow-uptoourfirst
study of the conformational changes occurring upon p47phox
activation (21). Here we precisely localized unmasked regions
during AIR and PX release. Indeed, the bottom of the tandem
SH3s groove, including the Src loops extending from both
SH3s, becomes accessible. This is a definitive structural dem-
onstration that the addition of targeted negative charges
induces AIR release (by mutation here and phosphorylation in
vivo). At this point, p47phoxis now able to progress further to
the next step along the activation process: the interaction with
p22phox.
The existence of a “dormant” state of p47phox, involving
region unmasking upon activation, was proposed 16 years ago
(13). Since then, the two SH3s domains and the N-terminal
region of the protein (designated as the PX domain in 1996)
have been pointed out in this mechanism (34, 35). The PX
domain lipid binding property was identified in 2001 (10), and
its enhancement in the activated form clearly established 2
years later (8). Over this long period, the PX domain was
believedtointeractinternallywithSH3B(4,7,8,16,24,30).The
structureofthetandemSH3s-AIRsupercomplexraiseddoubts
about this interaction (17–19). Finally, small angle x-ray scat-
tering molecular envelopes of the entire p47phoxinstead sug-
gested that SH3A was the internal PX target (20). Our present
studyrevealstheunmaskingofanewsurfaceonthelateralside
ofSH3A.Thisenhancedaccessibilityandthehigherlipidbind-
ingpropertyoftheactivatedstate(Fig.6C)stronglysupportthe
proposal that this lateral surface can be involved in PX
interaction.
Identification of Residues Involved in PX-SH3A Interaction
by Site-directed Mutagenesis—To more precisely identify the
residues that are directly involved in PX binding, five mutants
werecreatedwithinthenewsurfaceidentifiedbyDXMS.None
of them showed interaction with the p22phoxCter part (Fig. 6, A
and B, and data not shown), confirming tandem SH3s-AIR
supercomplex integrity.However,
p47phoxR162A/D166A-?Cter was able to interact with specific
phospholipids (Fig. 6C), reflecting PX domain release. The sin-
glemutantsR162AandD166Adonotshowsuchenhancement
in lipid binding (Fig. 6C), showing a synergistic effect of both
residues in PX domain locking. Interestingly, the 2 residues
identifiedarelocatedwithinpeptides161–166,whichshowthe
largest deuteration difference between p47phox?Cter and
p47phoxTM-?Cter in DXMS local kinetics (Fig. 4B). This sug-
gests that these residues play a role in the p47phoxautoinhibi-
tion mechanism. This semi-open conformation is further sup-
ported by DXMS global kinetics showing an intermediate
surface accessibility between the inhibited and activated states
for p47phoxR162A/D166A-?Cter.
Arecentstudypointedoutthattheregionupstreamfromthe
SH3A module (residues 151–158) could be involved in the lip-
id-binding site inhibition, as shown by the PX domain opening
upon substitution by a polyglycine linker (23). Another study
the doublemutant
showed that residues Ile-152 and Thr-153 are crucial for Nox2
activation (36) but do not affect p47phoxtranslocation proper-
ties in response to phorbol myristate acetate addition. These
twostudiesunderlinearoleforthisstretchofaminoacidslocal-
ized close to the surface highlighted here by DXMS. However,
this stretch is already fully deuterated after 30 s, and no deu-
teration difference can be seen between p47phoxactivated and
autoinhibited forms (Fig. 3C). These DXMS data firstly prove
that this 151–158 region is readily accessible. Secondly, the ab-
sence of deuteration difference between both states does not
allow conferring on this region any structural role in p47phox
activation. It may be involved at a later stage, after oxidase
assembly, as suggested by Taura et al. (36).
Intramolecular Signal Transduction within p47phoxupon
Activation, Coupling between AIR Release and PX Opening—
Apart from identifying the PX docking site on SH3A, an even
more important pursuit is understanding the domino effect
explaining how phosphorylations occurring in the p47phox
C-terminalpartmakethePtdIns(3,4)P2-bindingsiteaccessible,
threemodulesupstreamalongtheprotein.Amongthedifferent
mutants studied here, the properties of the double mutant
R162A/D166A address this question.
WhenlookingcloseratthetandemSH3s-AIRsupercomplex
structure (18, 19), residue Arg-162 from SH3A appears to be
directlyconnectedtotheAIR(Fig.7A).Arg-162isatthecenter
ofahydrogen-bondnetworkconnectingtheSH3Asurfacewith
the AIR in its bound state (Fig. 7B). It interacts directly with
His-309 and indirectly with Ser-310 through stabilization of
residueGlu-211.FromthiscorenetworkaroundArg-162,addi-
tional residues Ile-164 and Pro-212 from the SH3A and Ile-311
and His-312 from the AIR are involved in additional contact
within this interface (Fig. 7B).
In contrast, no interaction of residue Asp-166 with the AIR
canbestatedfromtheavailablecrystalstructures(ProteinData
Bank (PDB) 1NG2 and 1UEC). Therefore, the synergistic effect
observedinthedoublemutantcanbeexplainedbyacombined
stabilization of the PX domain by residues Arg-162 and Asp-
166. Mutations of these residues lead to PX domain release
without affecting tandem SH3s-AIR supercomplex integrity,
but AIR release (such as in p47phoxTM-?Cter) also leads to PX
domain release.
The coupling between AIR and the PX domain can be
assigned to Arg-162. Upon AIR release, triggered by serine
phosphorylations, disruption of the hydrogen-bond network
aroundArg-162willdisorganizethesidechainstructuralorga-
nization of the highlighted peptides 162–166. This could be
sensed by the PX domain docked on this lateral surface and
would finally trigger its release (Fig. 7C). As already mentioned
byourgroup(21)andothers(8,16,23),althoughnotactivated,
p47phox?Cter shows a basal interaction with lipids in pulldown
experiments(Fig.6C).Thisreflectsanequilibriumbetweenthe
twodifferentPXconformations,closedandopen.Inthecaseof
p47phoxTM-?Cter,becausetheAIRisreleased,thePXdocking
site is destabilized, particularly around residue Arg-162, allow-
ing a displacement of this equilibrium that favors the open
form, i.e. the lipid-bound form. In light of the results presented
here, we can assume that in the case of p47phoxR162A/D166A-
?Cter, the PX is not only released but also unable to interact
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with the lateral surface of SH3A due to disappearance of
these crucial docking residues. In this mutant, the equilib-
rium is totally displaced toward the open form of the PX.
This explains the much higher interaction with lipids
observed for p47phoxR162A/D166A-?Cter than for p47phox
TM-?Cter (Fig. 6C).
A more systematic site-directed mutagenesis study must be
conducted on both regions encompassing residues 306–312
from the AIR and this newly identified surface from SH3A to
describe this mechanism more accurately. Mutations in the PX
domain could also lead to the identification of the residues
involved in the interaction with this SH3A lateral surface.
For many years, researchers have been trying to explain how
the phosphorylation of at least 3 serine residues on one part of
p47phoxleadstothePXdomainrelease.Hereasurfaceinvolved
inPXautoinhibitionhasfinallybeenlocalizedonSH3A.More-
over,2residuesfromthisregionarepointedoutasplayingakey
role in the intramolecular signal transduction from the AIR to
the PX domain. The emerging mechanism deciphered here in
p47phoxto control a global assembly process in response to
phosphorylations has never been reported before. To our
knowledge, it is a unique structural organization enabling an
internal signal transduction along four distinct modules.
Acknowledgments—We thank Daniel Kavan for developing the
scripts, thus facilitating data processing and interpretation. We also
thankPatriceVachetteandDominiqueDurandforcarefulreadingof
the manuscript and fruitful scientific discussions.
REFERENCES
1. Sumimoto, H. (2008) FEBS J. 275, 3249–3277
2. Bedard, K., and Krause, K. H. (2007) Physiol. Rev. 87, 245–313
3. Segal, A. W. (1996) Mol. Med. Today 2, 129–135
4. Lapouge, K., Smith, S. J., Groemping, Y., and Rittinger, K. (2002) J. Biol.
Chem. 277, 10121–10128
5. Nakamura, R., Sumimoto, H., Mizuki, K., Hata, K., Ago, T., Kitajima, S.,
FIGURE 7. Molecular mechanism of PX release upon AIR phosphorylation. A, structure of the tandem SH3s inhibited by the AIR (PDB: 1NG2) (18). SH3A,
SH3B,andAIRarerepresentedingreen,yellow,andorange,respectively.ResiduesArg-162,Asp-166,andHis-309areshownassticks.B,close-upshowingthe
network of polar interactions between residues Arg-162, Ile-164, Glu-211, and Pro-212 from SH3A (green) and residues His-309, Ser-310, Ile-311, and His-312
from AIR (orange). C, schematic representation of p47phoxactivation mechanism showing how phosphorylation of the AIR leads to the PX domain release
through the surface identified in this study.
Decipheringthep47phoxActivationMechanism
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at CNRS, on September 3, 2010
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http://www.jbc.org/content/suppl/2010/06/30/M110.139824.DC1.html
Supplemental Material can be found at:

Page 11

Takeshige, K., Sakaki, Y., and Ito, T. (1998) Eur. J. Biochem. 251, 583–589
6. Noda, Y., Kohjima, M., Izaki, T., Ota, K., Yoshinaga, S., Inagaki, F., Ito, T.,
and Sumimoto, H. (2003) J. Biol. Chem. 278, 43516–43524
7. Massenet, C., Chenavas, S., Cohen-Addad, C., Dagher, M. C., Brandolin,
G., Pebay-Peyroula, E., and Fieschi, F. (2005) J. Biol. Chem. 280,
13752–13761
8. Ago, T., Kuribayashi, F., Hiroaki, H., Takeya, R., Ito, T., Kohda, D., and
Sumimoto, H. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4474–4479
9. Hiroaki, H., Ago, T., Ito, T., Sumimoto, H., and Kohda, D. (2001) Nat.
Struct. Biol. 8, 526–530
10. Kanai,F.,Liu,H.,Field,S.J.,Akbary,H.,Matsuo,T.,Brown,G.E.,Cantley,
L. C., and Yaffe, M. B. (2001) Nat. Cell Biol. 3, 675–678
11. Stahelin, R. V., Burian, A., Bruzik, K. S., Murray, D., and Cho, W. (2003)
J. Biol. Chem. 278, 14469–14479
12. Zhan, Y., Virbasius, J. V., Song, X., Pomerleau, D. P., and Zhou, G. W.
(2002) J. Biol. Chem. 277, 4512–4518
13. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y.,
Ohno, M., Minakami, S., and Takeshige, K. (1994) Proc. Natl. Acad. Sci.
U.S.A. 91, 5345–5349
14. Taylor, R. M., Lord, C. I., Riesselman, M. H., Gripentrog, J. M., Leto, T. L.,
McPhail,L.C.,Berdichevsky,Y.,Pick,E.,andJesaitis,A.J.(2007)Biochem-
istry 46, 14291–14304
15. Wilson, L., Butcher, C., Finan, P., and Kellie, S. (1997) Inflamm. Res. 46,
265–271
16. Karathanassis, D., Stahelin, R. V., Bravo, J., Perisic, O., Pacold, C. M., Cho,
W., and Williams, R. L. (2002) EMBO J. 21, 5057–5068
17. Nauseef, W. M. (2004) Histochem. Cell Biol. 122, 277–291
18. Groemping, Y., Lapouge, K., Smerdon, S. J., and Rittinger, K. (2003) Cell
113, 343–355
19. Yuzawa, S., Suzuki, N. N., Fujioka, Y., Ogura, K., Sumimoto, H., and Ina-
gaki, F. (2004) Genes Cells 9, 443–456
20. Durand,D.,Cannella,D.,Dubosclard,V.,Pebay-Peyroula,E.,Vachette,P.,
and Fieschi, F. (2006) Biochemistry 45, 7185–7193
21. Marcoux, J., Man, P., Castellan, M., Vive `s, C., Forest, E., and Fieschi, F.
(2009) FEBS Lett. 583, 835–840
22. Zhang, Z., and Marshall, A. G. (1998) J. Am. Soc. Mass Spectrom. 9,
225–233
23. Shen, K., Sergeant, S., Hantgan, R. R., McPhail, L. C., and Horita, D. A.
(2008) Biochemistry 47, 8855–8865
24. Ago, T., Nunoi, H., Ito, T., and Sumimoto, H. (1999) J. Biol. Chem. 274,
33644–33653
25. Tang, J. (1963) Nature 199, 1094–1095
26. Zhang,H.M.,Kazazic,S.,Schaub,T.M.,Tipton,J.D.,Emmett,M.R.,and
Marshall, A. G. (2008) Anal. Chem. 80, 9034–9041
27. Yuzawa, S., Ogura, K., Horiuchi, M., Suzuki, N. N., Fujioka, Y., Kataoka,
M.,Sumimoto,H.,andInagaki,F.(2004)J.Biol.Chem.279,29752–29760
28. Nobuhisa, I., Takeya, R., Ogura, K., Ueno, N., Kohda, D., Inagaki, F., and
Sumimoto, H. (2006) Biochem. J. 396, 183–192
29. Ogura, K., Nobuhisa, I., Yuzawa, S., Takeya, R., Torikai, S., Saikawa, K.,
Sumimoto, H., and Inagaki, F. (2006) J. Biol. Chem. 281, 3660–3668
30. Shiose, A., and Sumimoto, H. (2000) J. Biol. Chem. 275, 13793–13801
31. Sumimoto,H.,Hata,K.,Mizuki,K.,Ito,T.,Kage,Y.,Sakaki,Y.,Fukumaki,
Y., Nakamura, M., and Takeshige, K. (1996) J. Biol. Chem. 271,
22152–22158
32. Gorzalczany, Y., Sigal, N., Itan, M., Lotan, O., and Pick, E. (2000) J. Biol.
Chem. 275, 40073–40081
33. Li, X. J., Fieschi, F., Paclet, M. H., Grunwald, D., Campion, Y., Gaudin, P.,
Morel, F., and Stasia, M. J. (2007) J. Leukoc. Biol. 81, 238–249
34. Leto, T. L., Adams, A. G., and de Mendez, I. (1994) Proc. Natl. Acad. Sci.
U.S.A. 91, 10650–10654
35. Ponting, C. P. (1996) Protein Sci. 5, 2353–2357
36. Taura,M.,Miyano,K.,Minakami,R.,Kamakura,S.,Takeya,R.,andSumi-
moto, H. (2009) Biochem. J. 419, 329–338
Decipheringthep47phoxActivationMechanism
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