Effects of p47phox C Terminus Phosphorylations on Binding Interactions with p40phox and p67phox

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 Å resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

The neutrophil NADPH oxidase is a central component of the nonspecific host resistance against microbial infection. The critical role of this enzyme is illustrated by a genetic disease, chronic granulomatous disease (CGD), 1 in which mutations in genes coding for NADPH oxidase components impair the ability of the patient to fight against infection. The NADPH oxidase is composed of several proteins, a membrane-bound heterodimeric flavocytochrome b (gp91 phox and p22 phox ), cytosolic proteins (p40 phox , p47 phox , p67 phox ), and a small GTP-binding protein (Rac1 or Rac2) (for review, see Ref. 1). Upon exposure to specific stimuli, activation of the enzyme takes place through multiple phosphorylation events of the cytosolic components (2)(3)(4)(5). Phosphorylations rearrange the protein-protein interactions among the cytosolic proteins ending with their translocation, simultaneously with Rac-GTP, to the membrane embedded flavocytochrome b. The resulting activated complex catalyzes the electron transfer from NADPH to O 2 leading to the production of superoxide anions, thus generating highly reactive oxygen species (ROS) in the phagocytic vacuole. For many years these ROS have been designated as the main actors in the neutrophil killing activity. Recent work suggests that in addition to its role in ROS formation, O 2 . generation leads to phagosomal Cathepsin G and Elastase mobilization in the microbicidal process (6). The three cytosolic proteins p47 phox , p67 phox , and p40 phox are modular proteins formed by various domains, such as SH3, PX, PB1, and TPR, usually found in signal transduction cascades. Numerous possible interactions within these proteins have been reported and are summarized in Fig. 1A. p47 phox and p67 phox are known to be essential for activation. In CGD patient lacking p47 phox , p67 phox is not translocated to the membrane. In contrast p67 phox is not essential to p47 phox translocation (7). In the activation process, p47 phox is the sensor of the activation signal through multisite phosphorylations and secondly the scaffolding protein conducting translocation of the other cytosolic factors to the membrane component of the oxidase. p67 phox is an activating factor in the electron transfer reaction (8). In contrast to p47 phox and p67 phox , nowadays no CGD related to p40 phox is known. p40 phox was first identified as a p67 phox -associated protein in resting neutrophils (9 -11). p40 phox translocates to the membrane upon stimulation in a p47 phox -dependent manner (12). The protein comprises the fol-lowing domains, PX, SH3, and PB1 ( Fig. 1A) (13). These modules confer to p40 phox the ability to interact respectively with phosphatidylinositol 3-phosphate (14 -16), p47 phox C terminus (17)(18)(19), and p67 phox PB1 domain (20). The physiological role of p40 phox is still a matter of debate. It has been postulated to modulate superoxide production, either positively (21)(22)(23) or negatively (18, 24 -26). In the last couple of years, an increasing body of structural and biochemical data shed light on the organization of the cytosolic complex in the resting state and on some events consecutive to p47 phox phosphorylation. In the resting state p47 phox , p67 phox , and p40 phox are thought to exist as a 1:1:1 ternary complex (27) as shown with purified proteins. Although p40 phox and p67 phox are both able to interact with the C-terminal polyproline region of p47 phox , the p47 phox /p67 phox complex is favored (19,27). A ternary complex can be achieved by the association of p40 phox with p67 phox (28) through their respective PB1 domain (20,(27)(28)(29) (Fig. 1B). However, it has been recently shown (30) that, in vivo, p47 phox is dissociated from the p67 phox /p40 phox complex, as suggested previously (31).
Activation initiated by the binding of chemotactic substances to neutrophil receptors leads, through signaling pathways, to primary phosphorylation of Ser-359 and/or Ser-370 of p47 phox (5). Additional phosphorylations can occur among the seven other C-terminal serines of p47 phox . Among them, Ser-303, Ser-304, Ser-328, and Ser-379 have been identified as important for membrane translocation, interaction with p22 phox , and the activation of the complex as shown by several mutagenesis studies (4,32,33).
It is now well established that these phosphorylations modify p47 phox binding properties. From the resting to the activated state, p47 phox is proposed to change from a closed to an open conformation where the PX domain is either masked by the internal C-terminal SH3 of p47 phox or free for interaction with lipids of the membrane (34,35). In addition, the two p47 phox -SH3 domains have been shown to be either buried or accessible to the p22 phox polyproline (32,36). Interactions with lipids and p22 phox are believed to account for the association of p47 phox with the membrane (37,38). Despite these new insights on p47 phox , there is almost no information concerning the reorganization of p67 phox and p40 phox in the cytosolic complex upon phosphorylation. When examining the extended possibilities of interactions between cytosolic factors (Fig. 1A) and the limited panel of those present in the resting state (Fig. 1B), a rearrangement involving the three proteins has been suggested (24,27). At the present stage, several structures of individual domains or of complexes made by two domains are available (16, 29, 34 -36, 39 -41). It has been proposed that the phosphorylation of p47 phox could modulate its interaction with p67 phox or p40 phox , respectively (17,24,27). To understand the structural rearrangements occurring upon activation, structural information on the effect of nonphosphorylated and phosphorylated serines is needed. We addressed this question, characterizing at a molecular level the interaction of the SH3 domain of p40 phox (SH3 p40 ) with the polyproline region of p47 phox .
We report the three-dimensional structure of SH3 p40 with and without the C-terminal polyproline of p47 phox at 1.46 and 2 Å resolution, respectively. Comparison with the structure of the C-terminal SH3 domain of p67 phox (C-SH3 p67 ) complexed to the p47 phox C terminus (41) highlights a strong difference between both SH3 domains. We compare the dissociation constants of both SH3 p40 and C-SH3 p67 with polyproline peptides by fluorometric techniques. The effect of phosphorylation on Ser-359, Ser-370, and Ser-379 on the binding of both SH3 toward p47 phox is also investigated.

Protein Expression and Purification
SH3 p40 -A DNA fragment encoding the p40 phox SH3 module, from Arg-174 to Lys-228, was amplified by PCR using the full-length p40 phox sequence (31) as a template. The PCR fragment was cloned into the pCRscript Amp SK(ϩ) cloning vector according to the manufacturer's protocol (Stratagene). The resulting pCRscript-SH3 p40 plasmid was cleaved with NdeI and XhoI and the 168-base fragment containing the SH3 p40 sequence was subcloned in pIVEX 2.4 (Roche Applied Science). The resulting vector named pIVEX-SH3 p40 was transformed and amplified in Escherichia coli BL21(DE3) strain. The sequence of the construct was checked by sequencing.
The His-SH3 p40 was overexpressed as described for p40 phox (19). After 3 h, cells were harvested and resuspended in chilled lysis buffer (20 mM Hepes, pH 7.5, 0.4 M NaCl supplemented with 40 mM imidazole and 5% glycerol). All of following operations were carried out at 4°C. Cells were disrupted by sonication then centrifuged at 40,000 rpm for 40 min in a Beckman 45 Ti rotor. The supernatant was loaded at 2 ml/min on 4 ml of Ni 2ϩ -nitrilotriacetic acid column equilibrated in the previous buffer. Proteins were eluted with a 50-ml linear imidazole gradient (40 -300 mM) at 2 ml/min. Fractions containing His-SH3 p40 were pooled, concentrated to 6 mg/ml, and subjected to overnight digestion at 20°C with 2.4 units of factor Xa/mg of protein in the presence of 2.5 mM CaCl 2 . Digestion products were loaded at a flow rate of 1 ml/min onto the Hiload 16/60 Superdex 75 gel filtration column (Amersham Biosciences) equilibrated in 20 mM Hepes, pH 7.5, 150 mM NaCl. The  (17, 18, 31, 37, 60 -65). B, interactions existing in vitro, in a resting state model, between nonphosphorylated recombinant cytosolic factors (20,27).
resulting protein fractions containing SH3 p40 were concentrated to 14 mg/ml. The final recombinant protein comprised 5 N-terminal residues (LIKHM) from the previous linker, as determined by N-terminal sequencing.
C-SH3 p67 -The sequence encoding residus 455-516, the C-terminal SH3 domain of p67 phox (C-SH3 p67 ), was amplified by PCR and cloned into pET15b (Novagen). The sequence of the resulting vector named, pET-CSH3 p67 , was checked by sequencing. The protein was expressed in E. coli BL21(DE3) strain and first enriched by affinity chromatography on a Ni 2ϩ -nitrilotriacetic acid column equilibrated in 20 mM Hepes, pH 7.5, 150 mM NaCl, 20 mM imidazole and eluted with a linear gradient of imidazole (20 -300 mM). The His tag was cleaved with thrombin (Sigma) overnight at 20°C with 2.4 units thrombin/mg of protein, and further purification was carried out by gel filtration on Superdex 75 (Amersham Biosciences) equilibrated with 150 mM NaCl, 20 mM Hepes, pH 7.5, 2 mM dithiothreitol.

Proline-rich region of p47 phox
The peptide KPQPAVPPRPSAD (residues 360 -372 of p47 phox ) was synthesized using conventional technology. The peptide was purified by HPLC using a C18 column and a linear gradient of 5-60% CH 3 CN in water with 0.1% trifluoroacetic acid. The peptide was lyophilized prior to resuspension in water (50 mg/ml) for crystallization. Peptide concentration was determined by amino acids analysis. The six other peptides were synthesized by Neosystem (Table II). They were repurified by HPLC using a C18 column and a linear gradient of 5-60% CH 3 CN in water with 0.1% trifluoroacetic acid. The purity of the peptides was higher than 95%.

Tryptophan Fluorescence Spectroscopy
Fluorescence measurements were performed at 20°C in a Fluoromax (Jobin-Yvon) spectrofluorometer. The excitation wavelength was set at 290 nm, and the fluorescence emission was recorded between 295 and 450 nm. Increasing amounts of peptide were added to a fluorescence cuvette containing 100 l of a SH3 p40 solution. The resulting shift of maximal emission wavelength (⌬) was monitored over a large range of p47 phox peptide concentration. The equilibrium dissociation constant (K d value) characterizing the SH3/polyproline interaction was determined according to Equation 1 under the assumption that the fluorescence shift ⌬ was proportional to the concentration of the SH3/polyproline complex.

Crystallization
Crystals were grown by hanging-drop vapor diffusion by mixing equal volumes of protein and reservoir solutions. The best diffracting crystals of free SH3 p40 were obtained at 14°C from a protein solution at 14 mg/ml in 20 mM Hepes, pH 7.5, 150 mM NaCl, and a reservoir solution containing 200 mM sodium acetate, pH 4.6, 100 mM ammonium sulfate, 40% polyethylene glycol monomethyl ether 2000. Crystals grew up within 2 months. Crystals of SH3 p40 /polyPro p47 were grown at 20°C with a reservoir containing 100 mM sodium citrate/citric acid, pH 5, 2.4 M ammonium sulfate. To obtain the complex, SH3 p40 at 14 mg/ml in 20 mM Hepes, pH 7.5, and 150 mM NaCl was mixed at 1:5 molar ratio to the polyproline peptide solution (50 mg/ml in water). This ratio was determined from the dissociation constant extrapolated from fluorescence measurements and corresponds to the presence of roughly 90% complexed SH3 p40 /polyPro p47 . Crystals were obtained within two or 3 weeks. Their size and quality were improved by macro-seeding with a reservoir solution containing 100 mM citrate/citric acid, pH 5, and 2.2 M ammonium sulfate to a final size of 200 ϫ 50 ϫ 20 m.
Prior to x-ray diffraction experiments crystals were flash frozen in liquid nitrogen. Free SH3 p40 crystals were frozen directly from the mother liquor and SH3 p40 /polyPro p47 crystals were soaked in a cryobuffer (reservoir solution containing 15% glycerol) prior to freezing.

Data Collection, Structure Determination, and Refinement
Free SH3 p40 -Free SH3 p40 crystals cooled at 100 K diffracted to 2 Å on a rotating anode (Nonius) using a MARCCD detector (Mar 345, X-ray Research). Crystals diffracted anisotropically and were highly mosaic; several crystals were screened to obtain the best diffracting crystal. Data were processed with DENZO and SCALEPACK (42) and reduced with the CCP4 package (43). Statistics are shown in Table I. The space group is pseudo-orthorhombic and R sym values are 11.5 and 10.8% in C222 1 or P2 1 space groups, respectively. The structure of SH3 p40 was solved by molecular replacement using the program AMORE (44). The search model was a superposition of eight SH3 structures: SEM (PDB code 1SEM), spectrin (PDB code 1SHG), tyrosine kinase Abl (PDB code 1ABQ), tyrosine kinase Fyn (PDB code 1FYN), Eps8 (PDB code 1I0C), Crk (PDB code 1B07), amphiphysin 2 (PDB code 1BB9), Hck (PDB code 1BU1), restricted to main chain atoms. These SH3 domains share 38,33,15,20,22,24,22, and 18% sequence identity with SH3 p40 , respectively. Molecular replacement using each structure individually as a starting model failed. The model of SH3 p40 was refined using CNS (45), and 5% of the data (randomly selected reflections) was excluded from refinement as free R-flagged reflections. The refinement was initially tried in both space groups C222 1 and P2 1 ; the R cryst values clearly excluded the orthorhombic form. The asymmetric unit contains two SH3 p40 domains, which were refined by constraining the core of the monomers (i.e. ␤-strands excluding the loops) with a 2-fold symmetry.
SH3 p40 /PolyPro p47 -The crystals flash frozen to 100 K diffracted poorly only showing elongated spots to 3.5 Å and a very high mosaicity that prevented data integration, despite the fact that no crystalline ice was formed in the liquid surrounding the crystal. To improve the diffraction, the crystal temperature was raised by blocking the cryostream for a few seconds and rapidly dropped back to 100 K. After a few cycles of annealing, crystals cooled to 100 K diffracted to high resolution. A complete data set to 1.46 Å was collected on beamline BM30A, European Synchrotron Radiation Facility-Grenoble, using a Mar CCD detector and a wavelength of 0.98 Å. The same programs as for the free SH3 p40 were used for data treatment; statistics are shown in Table I. The space group is P2 1 2 1 2 1 with a unit cell of a ϭ 39.6 Å, b ϭ 50.5 Å, c ϭ 68.2 Å. Free SH3 p40 was used as a search model to solve the structure of SH3 p40 /polyPro p47 by molecular replacement. SH3 p40 /polyPro p47 crystals contain two 1:1 complexes in the asymmetric unit. After a few cycles of refinement with CNS, the polyproline peptide was build automatically using the program ARP (46). During the refinement, no constraints between the molecules in the asymmetric unit were applied. Both structures, free SH3 p40 and SH3 p40 /polyPro p47 , were refined by iterative cycles of manual corrections with O (47) and energy minimization or simulated annealing followed by B-factor refinements using CNS. The models were refined to final R cryst and R free of 24.7 and 25.0% at 2 Å resolution for the SH3 p40 and R cryst and R free of 18.1 and 20.7% at 1.46 Å resolution for SH3 p40 /polyPro p47 . The quality of the final models was analyzed with Procheck (48) and Whatcheck (49).

SH3 p40
Structures-The crystalline arrangement of SH3 p40 crystals consists of tightly packed protein layers with weak connections in between layers that involve mainly C-terminal Lys-228 p40 from one monomer interacting with a symmetry related Lys-228 p40 from the second monomer. The weak connections in one direction (which is parallel to a-c) probably explain the anisotropy. The crystal packing for SH3 p40 / polyPro p47 is different and more compact. In both crystal forms, the two molecules present in the asymmetric unit are very similar with root mean square deviations on main chain atoms of 0.06 and 0.3 Å for SH3 p40 and SH3 p40 /polyPro p47 , respectively.
The free SH3 p40 structure comprises residues 174 -228 for both monomers present in the asymmetric unit and 61 water molecules. The SH3 p40 /polyPro p47 structure consists of residues 169 -228 for both SH3 p40 (chains A and B) and of residues 360 to 372 (chain C) and 360 -369 (chain D) for the polyPro p47 peptides associated to monomers A and B, respectively. PolyPro p47 peptides are acetylated in their N-terminal end. In addition to the protein, four sulfate ions, two trifluoroacetic acid molecules and 227 water molecules per asymmetric unit were located from the electron density maps and refined. The two SH3 p40 structures, free or in complex with polyPro p47 , are very similar even in the peptide binding site. Because of the much higher resolution of SH3 p40 /polyPro p47 crystals, this structure is discussed below.
The general topology of SH3 p40 is very similar to that of other SH3 domains (50 -53) and consists of five ␤-strands arranged as two orthogonal ␤-sheets, forming a compact antiparallel ␤-barrel ( Fig. 2A). The binding site located at the surface of the barrel is flanked by the RT and n-Src loops. These loops are variable among SH3 domains and contribute to the specificity of polyproline binding.
Polyproline Binding Site-Residues 360 -369 of polyPro p47 (360 p47 to 369 p47 ) are well determined in both molecules present in the asymmetric unit. They adopt a polyproline helix of type II (PPII) conformation with three residues per turn. The peptide is located in a groove formed by hydrophobic residues highly conserved among SH3 domains and negative electrostatic patches on the SH3 p40 surface ( Figs. 2A and 3A). Residues 360 p47 to 369 p47 interact with SH3 p40 mainly via van der Waals contacts involving Phe-179 p40 , Asn-184 p40 , Asp-206 p40 , Trp-207 p40 , Pro-220 p40 , and Phe-223 p40 located in the hydrophobic pocket of SH3 p40 (Fig. 4). A few hydrogen bonds are also involved, in particular between the indole nitrogen of Trp-207 p40 and the carbonyl oxygen of Pro-367 p47 or between the N⑀ of Gln-362 p47 and the carbonyl oxygen of Asp-180 p40 . Arg-368 p47 seems to play a key structural role in the complex as it is involved in several interactions: an electrostatic bonding with Glu-188 p40 located in the RT loop of SH3 p40 and van der Waals contacts involving the aliphatic side chain of Arg-368 p47 and Trp-207 p40 (Fig. 2B). Two water molecules are also involved in the peptide binding and bridge Ser-222 p40 to Ala-364 p47 and Asn-204 p40 to Pro-367 p47 . As a result of the complex formation, Trp-207 p40 is buried and hidden from the solvent in SH3 p40 /polyPro p47 in contrast to the free SH3 p40 .
Comparison of SH3 p40 /PolyPro p47 and C-SH3 p67 /p47 phox -Cter-SH3 p40 shares 44% sequence identity with C-SH3 p67 (Fig. 3E), and the structures superimpose with root mean square deviations on main chain atoms of less then 1 Å. The structure of C-SH3 p67 was solved by NMR in complex with the C-terminal end of p47 (residues 359 -390, PDB code 1k4u) (41). Fig. 3 compares the electrostatic surfaces of both SH3s showing the large negative surface for C-SH3 p67 due the presence of many acidic residues. In our structure the side chain of Arg-368 p47 has a low B-factor indicative of a very well defined position in the complex, in line with a strong electrostatic interaction with Glu-188 p40 . Negatively charged surfaces are limited to Glu-188 p40 located in the SH3 p40 RT loop (Fig. 3, A and C). On the contrary, in C-SH3 p67 /p47 phox -Cter (41), the side chain of Arg-368 p47 has multiple conformations as seen from the various NMR structures. In contrast, other residues in the vicinity of Arg-368 p47 presented only one side chain conformation. The C-SH3 p67 RT loop contains up to five acidic amino acids responsible for a strong negatively charged surface and allowing multiple positioning of the Arg-368 p47 side chain (Fig.  3, B and D). The superposition of both SH3 structures also highlights possible interactions of SH3 p40 with the C-terminal end of p47 phox beyond polyPro p47 residues present in our structure. Previously some residues of C-SH3 p67 were identified as part of the binding surface using NMR cross-saturations experiment (41). As seen in sequence alignment of both SH3 (boxes in Fig. 3E), all these residues are highly conserved (77% identity) or closely related in SH3 p40 . Ile-505 p67 and Val-490 p67 were identified as being particularly important in the interaction (gray boxes in Figs. 3E and Fig. 5).
Several serine residues in p47 phox -Cter have been shown to be critical in NADPH oxidase activation. Among the serine C-SH3 p67 . The surface is highly negative. The difference of Arg-368 p47 binding mode is highlighted in C and D. SH3 side chains of mainly acidic residues are shown in yellow and Arg-368 p47 is in orange. C, SH3 p40 /polyPro p47 (this work). D, a sample of various orientations from the different NMR structures is shown for Arg-368 p47 . E, sequence alignment between SH3 p40 and C-SH3 p67 . Residues of C-SH3 p67 involved in the p47 phox -Cter interaction (41) are boxed. Moreover, gray boxes underline residues Ile-505 p67 and Val-490 p67 directly in contact with non-PXXP motif of p47 phox -Cter. Residues are referred as identical (*), strongly similar (:), or weakly similar (.). Surfaces A and B were drawn with GRASP (66). C and D were drawn with PyMol.

FIG. 5. Interaction interface of the non-PXXP motif of p47 phox -Cter with C-SH3 p67 .
The surface of C-SH3 p67 is drawn in white and p47 phox -Cter in green. The complex used is the most representative structure from PDB file 1k4u (41). Side chain residues of C-SH3 p67 are in yellow. The corresponding residues of SH3 p40 are indicated in parentheses. The figure was drawn with PyMol. residues studied herein, only Ser-370 p47 is present in our structure. Its side chain points toward the solvent and is clearly not involved in the interaction with SH3 p40 . From 1k4u coordinates, the same observation is made for C-SH3 p67 . Ser-359 p47 and Ser-379 p47 are not present in our peptide, their interaction is extrapolated from the superposition of 1k4u coordinates with our structure. Ser-359 p47 is located at the N-terminal end of the peptide, its location is close to Glu-480 p67 equivalent to Ala-193 p40 . Ser-379 p47 interacts with Glu-496 p67 (conserved in SH3 p40 ) and from our extrapolated structure is likely to interact also with SH3 p40 .
Interaction between p47 phox and p40 phox or p67 phox Characterized by Fluorescence Measurements-We have measured the dissociation constants of a series of peptides corresponding to the PXXP motif (358 -372) or the whole p47 phox -Cter (358 -390) with SH3 p40 or the C-SH3 p67 (Fig. 6 and Table II). Both types of peptides (short or long) are almost identical to the peptides used for the structural studies and therefore they will be denoted in the following text polyPro p47 and p47 phox -Cter, respectively. The entire proteins, p47 phox and p40 phox , interact with an estimated K d of 5 M (19,27). This value is 40 times lower than the K d between SH3 p40 and polyPro p47 , suggesting that the interaction between the two proteins involves other regions in addition to the SH3/polypro couple. The K d of p47 phox -Cter toward SH3 p40 is 380 times lower than that of PolyPro p47 alone. Thus, our measurements show that the binding between SH3 p40 and p47 phox -Cter implicates both the PXXP motif and a non-PXXP region of p47 phox -Cter. Interestingly, it corresponds to the same interacting regions for p47 phox with C-SH3 p67 as identified by Kami et al. (41). In addition, our K d value for the C-SH3 p67 /p47 phox -Cter interaction (Table II) is consistent with their results. Although the same regions of p47 phox seem to be involved in the interaction with both SH3, it is clear from Table  II that the complex with C-SH3 p67 is favored (K d ratio of 11, line 4 in Table II). The preference for C-SH3 p67 is already observed for the short polyPro p47 peptide (K d ratio of 20, line 1 in Table II), and the additional residues of p47 phox -Cter do not modify significantly the difference in affinity toward SH3 p40 and SH3 p67 (comparison of line 1 and line 4 in Table II). Therefore, the contribution of the strict PXXP motif by itself induces the preference for p67 phox . The difference in affinities of p47 phox -Cter for both SH3 domains obtained from our fluorescence measurements is consistent with our structural data. Indeed, the comparison of the structures highlights a strong difference in the electrostatic potential surface between the two SH3 along the polyPro binding site that enhances the binding of polyPro p47 to C-SH3 p67 , whereas only few differences are present among the interactions with the extra C-terminal residues.
Effect of Ser-359/Ser-370 or Ser-379 Phosphorylation on the Binding of p47 phox to SH3 p40 and C-SH3 p67 - Table III summarizes data from the literature on the specific role of each serine phosphorylation. We have investigated the role of the phosphorylatable serines surrounding polyPro p47 on SH3 recognition. Various peptides have been synthesized, in which Ser-359 p47 and Ser-370 p47 are either substituted by aspartates or phosphorylated (Table II). Both modifications (mutation or phosphorylation), in either short or long peptide, do not affect significantly the binding to SH3 p40 . Therefore, as a first approximation, our observations validate the Ser-to-Asp mutation approach which has been widely used to mimic serine phosphorylations (4,5,32). The affinity of C-SH3 p67 for short polyPro p47 peptides is moderately reduced by aspartate mutations and slightly more by phosphorylations. Even in the absence of a direct interaction of Ser-359 p47 and Ser-370 p47 with C-SH3 p67 , the higher electronegative character of additional phosphates compared with Asp residues, added to the strong electronegative surface of C-SH3 p67 , might explain this effect. However, even if phosphorylation reduces the K d ratios toward both SH3 from 20 to 5 (comparison of lines 1 and 3 in Table II), C-SH3 p67 still has a stronger affinity than SH3 p40 . With p47 phox -Cter, in a native form or with Ser-to-Asp mutations, no major differences are observed, probably because the increase of binding interface with the addition of the non-PXXP region of p47 phox masks the small differences in affinity observed among the short peptides.
To address the question of the role of Ser-379 p47 during activation, binding properties of p47 phox -Cter, with Ser-379 p47 phosphorylated or not, have been investigated. Upon phosphorylation, the K d ratios of the two peptides toward both SH3s increase by a factor of 5 and 30 for SH3 p67 and SH3 p40 , respectively (comparison of lines 4 and 6 in Table II), denoting an important modification of the interaction between p47 phox -Cter and both SH3.
We show herein that phosphorylations of Ser-359 p47 and Ser-370 p47 have no significant impact neither on the binding of p47 phox -Cter to both SH3s nor in modifying the binding preference between SH3 p67 and SH3 p40 . The side chain orientation of the two serines in the structures explains the low effect of the phosphorylation of Ser-359 p47 and Ser-370 p47 ; both side chains point away from the interaction. In contrast, the phosphorylation of Ser-379 p47 destabilizes SH3 p40 /p47 phox -Cter and C-SH3 p67 /p47 phox -Cter with a stronger effect for SH3 p40 .

Structural Determinants of the Interaction between p47 phox -Cter and SH3 p40
Two possible SH3 interacting partners have been identified for the polyproline motif of the p47 phox C terminus: SH3 p40 and C-SH3 p67 (Fig. 1A). Various models for the cytosolic complex in the resting state have been proposed, arguing for polyPro p47 complex with either SH3 p40 or C-SH3 p67 (10,12,31). Several studies based on the respective affinities of both p40 phox and p67 phox for p47 phox (19,27,54,55) led to the accepted model of a trimeric organization (Fig. 1B) in which C-SH3 p67 interacts with polyPro p47 , while p40 phox and p67 phox are held together via a PB1/PB1 interaction (27). Despite the importance of SH3polyproline interactions in the NADPH oxidase complex, no high resolution structural information is available to explain the favored interactions occurring within the p47 phox /p67 phox / p40 phox complex. Previous NMR-based structure of C-SH3 p67 / polyPro p47 highlighted the contribution of the whole p47 phox C-terminal sequence, downstream the PXXP motif (41). Indeed, this additional non-PXXP sequence showed an extra interaction that results in high affinity and specificity. This suggested that the non-PXXP region can be responsible for stronger interactions with C-SH3 p67 rather than SH3 p40 . To discuss the role of the non-PXXP region of p47 phox in SH3 p40 /p47 phox , we compared the binding properties of p47 phox -Cter to the SH3 modules of p67 phox and p40 phox , based on fluorometric results and three-dimensional structure analysis. Fluorescence measurements reveal three major points: 1) as for C-SH3 p67 , SH3 p40 is able to establish an additional interaction with the non-PXXP motif of p47 phox -Cter, 2) the stronger affinity for C-SH3 p67 than for SH3 p40 is due to the PXXP motif itself and more precisely the Arg-368 p47 binding mode, and 3) the isolated SH3 p40 module presents a higher affinity for p47 phox -Cter than the whole p40 phox protein for p47 phox .
p40 phox Interacts with a PXXP and a Non-PXXP Motif of p47 phox -Cter-SH3 p40 exhibits a K d of 0.52 M for p47 phox -Cter, which is a high affinity compared with 10 -50 M for most SH3-peptide interactions. Using p47 phox peptides corresponding to the PXXP motif (residues 358 -372) or corresponding to p47 phox -Cter (residues 358 -390), we show that the C-terminal non-PXXP region is not responsible for the difference in affinities toward the two SH3 modules. Despite the fact that the C-terminal region allows in each case a much stronger affinity than with the PXXP motif alone, the specificity resides mainly within the PXXP motif recognition. Interaction of both SH3 with the non-PXXP motif of p47 phox -Cter seems to be identical, the residues of C-SH3 p67 identified as part of the binding surface for this noncanonical interaction have been previously identified using NMR cross-saturation experiments (Fig. 3E) (41).
Stronger Affinity of p47 phox -Cter toward SH3 p67 Is Explained by the Arg-368 p47 Binding Mode-Interactions of polyPro p47 with both SH3 are mainly hydrophobic except for Arg-368 p47 . A strong difference in the electrostatic potential surface of the two SH3 may be responsible for the stronger binding of PXXP motif to C-SH3 p67 . Indeed, Arg-368 p47 interacts only with Glu-188 p40 in our structure, whereas it has multiple interacting mode with the RT loop of the C-SH3 p67 . Previous experiments have focused on the importance of Arg-368 p47 in the binding strength (41): (i) replacement of this arginine by an alanine decreases affinity for C-SH3 p67 by a factor of 625 and (ii) binding of the isolated non-PXXP motif of p47 phox (residue 369 p47 to 390 p47 ) is observed only when Arg-368 p47 is added to the peptide. A much stronger negatively charged RT loop in C-SH3 p67 than in SH3 p40 can account for a higher association of the two binding partners and/or a stronger efficiency in keeping the protein complexed.
Modules Flanking SH3 p40 Modulate Its Affinity for p47 phox -Our fluorescence measurements reveal that the affinity between p47 phox and p67 phox is identical to the affinity between p47 phox peptides and C-SH3 p67 domain (Table II). This confirms that the p47 phox -p67 phox complex is completely determined by C-SH3 p67 and p47 phox -Cter sequences. On the contrary, strong difference in affinity between p47 phox and p40 phox on one side and between p47 phox -Cter and SH3 p40 on the other side is observed (Table II). The presence of PX and PB1 domains on each side of SH3 p40 lowers the affinity toward p47 phox by 1 order of magnitude. Thus, in p40 phox , the binding properties of SH3 p40 are tuned down by the flanking modules. Interestingly, Lopes et al. (26) have shown recently that phosphorylation of p40 phox on threonine 154 changes the conformation of the protein thus resulting in an inhibitory effect on NADPH oxidase. Thr-154 p40 is located between the PX and the SH3 domains (26), and the authors proposed that, after its phosphorylation, SH3 p40 could be exposed and compete with p67 phox for p47 phox binding. Moreover, Sathyamoorthy et al. (24) reported  Wildtype that transient expression of the SH3 p40 domain in vivo inhibits more efficiently the NADPH oxidase than the full-length p40 phox . Although the role of p40 phox (inhibitory or stimulatory) is still a matter of debate, these observations are in line with our results of an increased affinity toward p47 phox of SH3 p40 alone with respect to the whole p40 phox . However, such an increase in affinity due to a possible exposure of SH3 p40 , is not sufficient to compete with the strength of the p67 phox /p47 phox interaction (Table II). Additional effects have to occur that might be related to additional phosphorylations on p67 phox (56,57), p40 phox (58), and particularly p47 phox , which is polyphosphorylated (2, 3).

Phosphorylation on p47 phox C Terminus; Consequences toward the SH3/PolyPro Interaction between Cytosolic Factors
Phosphorylation on Ser-359/370 of p47 phox Does Not Promote a Switch of p47 phox Binding from C-SH3 p67 to SH3 p40 -Data from numerous studies delineating the phosphorylation of p47 phox serine residues during the activation cascade are summarized in Table III. Globally they suggest (5) that Ser-359/370 are phosphorylated first, allowing subsequent phosphorylation of Ser-379 for an efficient membrane translocation and finally of Ser-303/304 to induce the activity of the complex. The effect of Ser-303/304 phosphorylation, in the activation mechanism, has been clearly unraveled and confirmed (4,32,36) and involves the phosphorylation of Ser-328 p47 (32). Mutation of serines 359 and 370 to alanines abrogated completely the subsequent steps in the activation mechanism from consecutive phosphorylation on other serines to NADPH oxidase activation. In a cellular context, replacement of serines by glutamate or aspartate residues restored the phosphorylation events on other serines of p47 phox as well as membrane translocation of the cytosolic factors but not NADPH oxidase activity. Therefore prior phosphorylation of Ser-359/370 is necessary to allow subsequent phosphorylations and the activation cascade to take place (5). The presence of two phosphorylation sites, Ser-359/ 370, surrounding a polyproline motif crucial for the organization of the ternary complex suggested that phosphorylation may modify the interactions among the cytosolic factors during activation. Several groups suggested a possible switch from one SH3 to the other (24) during the phosphorylation process (17,27,58). At the molecular level, very little is known on the evolution of the interaction network between the cytosolic factors following phosphorylation. Until now studies have mainly focused on the phosphorylation consequences in the intramolecular organization of p47 phox itself. In our work, K d measurements of p47 phox peptides, phosphorylated or not, reveal no significant modifications in the difference in affinity toward the isolated SH3 domains of p67 phox and p40 phox . Thus, the switch hypothesis of binding of p47 phox from C-SH3 p67 to SH3 p40 , as a direct effect of phosphorylations on SH3 affinities, can now be ruled out.
Recent work (30) has shown that in the resting state before any priming event, p47 phox was dissociated from the ternary complex in the cytoplasm. The ternary complex was seen only in phorbol 12-myristate 13-acetate-activated neutrophils. These results suggest that in a cellular context, despite a possible strong interaction of p47 phox with the p67 phox /p40 phox heterodimer as observed in vitro (27), phosphorylations are required for ternary complex formation. Here we show that such phosphorylations are fully compatible with the formation of p47 phox -Cter/C-SH3 p67 complex, since they do not affect the dissociation constant. Thus, Ser-359/370 phosphorylation might promote the initial ternary complex formation in the neutrophil where the C terminus of p47 phox may not be acces-sible due to the cellular environment in contrast to recombinant cytosolic factors in solution.
Phosphorylation on Ser-379 p47 Weakens Cytosolic Complex in Vitro-Finally we investigated consequences of phosphorylation on Ser-379 p47 previously reported as a potential site of kinase action for NADPH oxidase regulation (2,33). The C-SH3 p67 /p47 phox -Cter structure solved by Kami et al. (41) shows that Ser-379 p47 is accessible and can contribute to the binding of the non-PXXP motif. Depending on the various NMR structures, Ser-379 p47 can establish an hydrogen bond with Glu-496 p67 , one of the residues identified in the interaction interface (Figs. 3E and 5). As seen in Table II, phosphorylation of Ser-379 p47 impairs the affinity of p47 phox -Cter for both SH3. This can be interpreted as a steric effect but more probably as a repulsive charge effect between the phosphorylated Ser-379 p47 and the neighboring residues Glu-496 p67 or Glu-209 p40 . The consequence of this phosphate addition in p47 phox -Cter is not SH3 specific and weakens the cytosolic factor complex. Ser-379 p47 was the first Ser whose mutation to Ala, reported in Table III, impaired oxidase activation and membrane translocation (33). The loss of affinity may be surprising for a translocation mechanism needing a tightly associated complex to co-migrate as a whole to the membrane for NADPH oxidase activation. However, upon Ser-379 p47 phosphorylation the modification of affinity toward SH3 p67 is moderate, contrary to the change of affinity toward SH3 p40 . The only conclusion that can be drawn from our study is that Ser-379 p47 phosphorylation will not promote SH3 p40 binding. The clarification of the cellular role of this serine phosphorylation will have to await further studies.
In summary, the p47 phox binding preference toward p67 phox rather than p40 phox is attributable to a negative contribution of SH3 p40 neighboring modules and the difference in the p47 phox polyPro binding properties is probably due to a strong difference in the negative net charge of the SH3 RT loops (Fig. 3). However, we show herein that SH3 p40 is competent for p47 phox binding with a strong affinity and specificity involving both PXXP and the non-PXXP motif of p47 phox -Cter. The existence of a physiologic situation in the activation or regulation mechanism where a SH3 p40 /p47 phox interaction takes place has not been evidenced so far. However, in light with the binding specificity of SH3 p40 toward p47-Cter, the reported roles of p40 phox in oxidase activity regulation could arise from a possible p40 phox /p47 phox interaction occurring during the various steps of the NADPH oxidase activation/deactivation mechanism. Moreover, we have shown clearly that the phosphorylation event on Ser-359/370 cannot initiate the activation cascade through modification of the SH3 binding properties and therefore the molecular mechanism involved here has still to be deciphered. Finally, we have shown that a phosphate on Ser-379 has a deleterious effect on the strength of interaction of p47 phox with SH3 p40 and C-SH3 p67 .
In addition to a better understanding of the molecular basis of the polyproline-SH3 interactions within the cytosolic phox complex, and a clarification of the phosphorylation consequence in the complexes formation and their stabilities, this work provides the high resolution structure of the last p40 phox module not yet reported. Indeed, the crystal structures of PX and PB1 domains have been previously reported isolated (16) or in complex with p67 phox PB1 (29). Thus, the whole structure of p40 phox is now known as individual pieces. Deeper understanding in the structure-function relationship of p40 phox will have to await three-dimensional positioning of the different modules with respect to each others. Moreover, a clear and definitive view of the kinetics and order of the phosphorylation events on p47 phox but also on p40 phox and p67 phox will be es-sential to solve the complete molecular puzzle of the activation cascade.