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1 Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
2 CREST, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
3 Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In the resting state, the oxidase subunits are localized separately at the membrane and cytoplasm. p47phox, p67phox and p40phox exist as a tight complex that can be purified by gel chromatography with an apparent molecular mass of 250–300 kDa (Wientjes et al. 1993; Park et al. 1994). Recently, the complex was found to form a ternary complex with 1 : 1 : 1 stoichiometry from sedimentation equilibrium studies (Lapouge et al. 2002). Upon activation of the cell, the ternary complex translocates from the cytosol to the plasma membrane and associates with flavocytochrome b558. This translocation process is controlled by a series of tightly regulated signalling events that assemble the NADPH oxidase subunits into the activated complex. p47phox has a key role in this process which contains a PX domain (Ponting 1996), tandem SH3 domains (N-SH3 and C-SH3) (Leto et al. 1990), a polybasic region/autoinhibitory region (PBR/AIR) (Sumimoto et al. 1994; Leto et al. 1994; Takeya et al. 2003) and a proline-rich region (PRR) (Finan et al. 1994) (Fig. 1A). In the inactive state, the binding surface of the tandem SH3 domains for the C-terminal cytoplasmic proline rich region of p22phox (p22phox PRR) is masked through an intra-molecular interaction with PBR/AIR, resulting in the autoinhibited form (Sumimoto et al. 1994; Leto et al. 1994; Ago et al. 1999; Huang & Kleinberg 1999). Anionic amphiphiles, activators of the NADPH oxidase in vitro, cause a conformational change in p47phox to expose its SH3 domains, resulting in binding to p22phox PRR and activation of the NADPH oxidase (Sumimoto et al. 1994; Swain et al. 1997; Shiose & Sumimoto 2000). Upon cell stimulation, some serine residues in PBR/AIR including Ser303, Ser304, Ser315, Ser320 and Ser328 are phosphorylated by protein kinase C (PKC) or Akt (El Benna et al. 1994, 1996; Fontayne et al. 2002; Hoyal et al. 2003). Mutational analyses have suggested that phosphorylation of Ser303, Ser304 and Ser328 were required for the NADPH oxidase activation (Inanami et al. 1998; Ago et al. 1999; Groemping et al. 2003). The phosphorylation of these Ser residues triggers conformational changes that subsequently lead to the rearrangement of the cytosolic components through intra- and inter-molecular interactions (Sumimoto et al. 1994; Ago et al. 1999; Huang & Kleinber 1999; Groemping et al. 2003). Thus, the tandem SH3 domains of p47phox are unmasked and are capable of binding to p22phox PRR (Sumimoto et al. 1994; Leto et al. 1994). This interaction is considered to be essential for the NADPH oxidase activation (Sumimoto et al. 1994, 1996; de Mendez et al. 1997).
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Results |
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The crystal of p47phox(151-340) contains one molecule in the asymmetric unit, which is an elongated monomer with an apparently partially unfolded structure. The elongated monomer is related to the other by a crystallographic 2-fold axis at the hinge to form a dimer that displays a dumbbell-like shape with a dimension of 89 x 64 x 64 Å. The monomers are intertwined with each other. This strand-exchanged and intertwined region was identified as the N-terminal SH3 domain although the distal loop of the canonical SH3 fold is extended to form the hinge.
The molecular mass of p47phox(151-340) used for crystallization was estimated to be 22 kDa by gel exclusion chromatography, showing that p47phox(151-340) exists as a monomer in solution. Furthermore, NMR studies and small-angle X-ray scattering analyses demonstrated that p47phox(151-340) has characteristic features of a monomeric globular protein in solution (our unpublished results). These results taken together support the notion that the intertwined dimer in the crystal is not physiologically relevant but could be stabilized in the crystal lattice. We could conclude that the half of the intertwined dimer split at the hinge represents the structure of p47phox(151-340) in solution, which we refer to as a globular module.
Recently, Groemping et al. (2003) also reported that the crystal structure of the residues 156-340 of p47phox reveals an intertwined dimer similar to our observation and concluded that the half of the intertwined dimer in the crystal structure is physiologically relevant. Here, we refrain from giving a detailed structural description of the intertwined dimer.
Structural characteristics of the globular module
In the globular module, the N-terminal SH3 domain (N-SH3, in blue), the linker (in orange), the C-terminal SH3 domain (C-SH3, in green) and the PBR/AIR (in pink) are readily identified as is expected from the domain structure of p47phox(151-340) (Fig. 1A,B). Both the SH3 domains take canonical SH3 folds, consisting of five anti-parallel ß-strands which form two ß-sheets packed at almost right angles and a single 310 helical conformation (Fig. 1B,C). The conserved ligand binding surfaces of N-SH3 and C-SH3 are arranged face to face and form a single binding groove (Fig. 1B). PBR/AIR starts with the 
A helix (residues 287–292) and the sequence RGAPPRRSSI (residues 296–305) follows. The sequence RGAPPRRSSI binds to the conserved binding surfaces of N-SH3 and C-SH3. Notably, the GAPPR sequence (residues 297–301) takes a left-handed polyproline type-II helix (PPII) that is simultaneously recognized by the hydrophobic groove formed by the binding surfaces of N-SH3 and C-SH3 (Fig. 1B). The N-terminal and the C-terminal flanking regions of the GAPPR sequence deviate from the classical PPII helical structure, where the presence of a short 310 helix (residues 302–304) following the PPII helix is noticeable. Then PBR/AIR leads to the coiled loop that runs to associate with N-SH3 and the linker. The remainder of PBR/AIR folds into the B helix (residues 321–331) that packs on C-SH3 so that C-SH3 is sandwiched between A and B. Novel recognition modes of PBR/AIR mediated by the two SH3 domains is a characteristic feature of p47phox(151-340).
Binding surfaces of the SH3 domains interact with PBR/AIR
Both SH3 domains could superpose with each other with an r.m.s. deviation of 1.3 Å for the C atoms of 50 aligned residues (Fig. 2A). The numerous entries within the Protein Data Bank (Berman et al. 2000) for the SH3 and SH3-like domains allowed extensive comparisons with the SH3 domains of p47phox(151-340). A structural comparison using the DALI search engine (Holm & Sander 1994) revealed that the N-SH3 domain from c-Crk (PDB code: 1CKA
[PDB]
) was the most closely related to the N-SH3 and C-SH3 domains of p47phox (a Z-score of 9.3 and an r.m.s. deviation between C atoms of 1.4 Å for 50 aligned residues of N-SH3 and a Z-score of 10.6 and an r.m.s. deviation between C atoms of 1.4 Å for 54 aligned residues of C-SH3). Superposition of the N-terminal SH3 domain of c-Crk with N-SH3 and C-SH3 of p47phox(151-340) is shown in Fig. 2A. The PPII binding surface is composed of a cluster of hydrophobic residues similar to the canonical PPII binding surface of the SH3 domains (Fig. 2A).
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The GAPPR sequence (residues 297–301) in PBR/AIR of p47phox(151-340) takes the PPII helix. It should be noted that the PPII helix is recognized by the SH3 domains simultaneously using the different sides as the basal planes, with the plus and minus orientations, respectively (Figs 2B,C and 3A). We will discuss the molecular recognition between these SH3 domains and the PPII helix in more detail below.
N-SH3 interacts with the RGAPPRRSSI sequence in PBR/AIR in the plus orientation (Fig. 2B). Although the GAPPR sequence is not the canonical sequence, it takes the PPII helix. Pro299 and Pro300 occupy the basal plane of the prism and bind to the hydrophobic pocket of the N-SH3 domain formed by Tyr167, Trp193, Pro206 and Phe209. This pocket is the typical PPII helix binding site of the SH3 domain and is generally referred to as P–1 and P0. While, Ala298 and Arg301 are located on the apex of the prism. Arg302, Ser 303 and Ser304 which follow the PPII helix form a 310 helix and Ile305 is now located in the pocket formed by Phe209 and Ala165. This pocket is formed by two aromatic residues in the canonical SH3 domain, and is referred to as P+2 and P+3, so that N-SH3 of p47phox is regarded as a variant with respect to the architecture of the binding pockets. Arg296 makes hydrophobic interactions with Trp193 but its interaction would not be stable, as the side chain of Arg296 shows a high B-factor (55.1 Å2). A set of two hydrogen bonds between the carbonyl group of Pro300 and the side chain of Ser208 and the carbonyl group of Gly297 and the side chain of Trp193 appears to be important for the stability of the backbone conformation of the GAPPR sequence (Figs 2B and 3A).
The C-SH3 domain recognizes the GAPPR sequence in the minus orientation (Fig. 2C). Pro299 and Ala298 bind to the hydrophobic pocket formed by Tyr237, Trp263, Pro276 and Tyr279. They are located in the bottom of the prism and occupy P–1 and P0, respectively. Pro300 and Gly297 are located on the apex of the prism formed by the PPII helix. Arg301 in the P–3 position makes hydrophobic interactions with Trp263 and Tyr274 and forms salt-bridges with Glu244 and Asp243 (Figs 2C and 3A). A set of two hydrogen bonds between the carbonyl oxygen of Pro299 and the side chain of Trp263 and between the amide nitrogen of Ala298 and the side chain of Tyr279 appears to be critical for the stability of the backbone conformation at the GAPPR sequence (Fig. 3A). The hydrophobic pockets corresponding to P+2 and P+3 in the C-SH3 domain are vacant (Fig. 2C).
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The ligand binding groove is formed by the conventional ligand binding surfaces of N-SH3 and C-SH3, and is occupied by the PPII helix formed by the GAPPR sequence. Two basal planes of the PPII prism are used as the interacting surfaces with N-SH3 and C-SH3 (Fig. 2B,C). One plane consisting of Gly297, Pro299 and Pro300 interacts with N-SH3, while the others, consisting of Ala298, Pro299 and Arg301, interacts with C-SH3, simultaneously where the side chain of Pro299 interacts with both the SH3 domains (Fig. 3A,B). These SH3 domains are related by a pseudo 2-fold symmetry axis at the centre of the binding groove so that the PPII helix interacts with N-SH3 and C-SH3 in a reverse orientation (Figs 1B and 3A). Thus, the ligand binding surfaces in both the SH3 domains are masked through intra-molecular interactions. Although the GAPPR sequence takes the PPII helix, the recognition by each SH3 domain is different from the canonical recognition. There is no PXXP motif or hydrophobic residues that confer specificity toward the SH3 domains. In this respect, GAPPR does not bind tightly to each SH3 domain, but the specificity of GAPPR is maintained by binding to both the SH3 domains, simultaneously.
Two SH3 domains are located close to each other around the n-Src loops (Fig. 3C), where the buried surface area between N-SH3 and C-SH3 is estimated to be 572 Å2. The interface involves two hydrogen bonds between the backbone carbonyl group of Glu190 in the n-Src loop of N-SH3 and the side chain of Ser277 at the first residue in the 310 of C-SH3 and between the backbone carbonyl group of Leu260 in the n-Src loop of C-SH3 and the side chain of Trp194 at the second residue in the ßCN of N-SH3 (Fig. 3D). In addition, there are several hydrophobic interactions between the side chains of Trp194 and Trp264 located in ßC and Leu260 and Glu190 in the n-Src loop, respectively. Gly192 and Gly262 at the tip of each n-Src loop are in close contact to maintain the proper orientation of the SH3 domains (Fig. 3C,D). It should be noted that there are missense mutations of the NCF-1 gene, the gene for p47phox in CGD patients, where Gly192 and Gly262 are substituted by Ser residues (Noack et al. 2001). These missense mutations could disrupt the orientation of the tandem SH3 domains. Indeed, the conformations of the n-Src loops in both the SH3 domains take rather compact structures, different from those of the canonical SH3 domains such as the N-terminal SH3 domain of c-Crk (Fig. 2A). The compact structures as well as the residues on the n-Src loop of both the SH3 domains seem to play a critical role in preventing a steric clash and maintaining the proper orientation.
The role of the linker in the autoinhibited form
The linker (residues 213–228) connecting N-SH3 and C-SH3 has a low average B-factor (29.8 Å2) similar to the SH3 domains, suggesting that the linker is involved in the structural core in the autoinhibited form. The N-terminal region of the linker (N-terminal linker) mainly interacts with N-SH3, while the C-terminal region of the linker (C-terminal linker) interacts with C-SH3, which are referred as site A and site B, respectively (Fig. 4A–C).
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In site B, several interactions between the C-terminal linker and C-SH3 which comprise hydrogen bonds and a salt bridge are dispersed on the surface of the C-SH3 domain (Fig. 4A,C). The side chain carbonyl group of Asp221 in the linker forms a hydrogen bond with the backbone amide groups of Leu260 in C-SH3, and the backbone carbonyl groups of Ile256 and Val255 in C-SH3 directly interact with the backbone amide protons of Asn225 and Glu229 in the linker, respectively. The backbone carbonyl group of Pro222 forms a hydrogen bond with the side chain of His257 and the side chain carboxyl group of Glu229 forms a salt bridge with the side chain of Lys258.
NOX1 is known to be a mammalian gp91phox homologue (Lambeth et al. 2000). Recently NOXO1/p41nox, the p47phox homologue, was identified and contains an N-terminal PX domain, tandem SH3 domains and a C-terminal conserved proline rich region (PRR) but lacks PBR/AIR (Banfi et al. 2003; Geiszt et al. 2003; Takeya et al. 2003) (Fig. 1C). Interestingly, the residues involved in the interaction, including Tyr161 in N-SH3, Asp217, Glu218, Asp221 and Glu229 in the linker, and Lys258 and His257 in C-SH3, are not conserved in NOXO1/p41nox (Fig. 1C). NOXO1/p41nox is constitutively active and does not need to form the autoinhibitory conformation. On the other hand, all of the above residues in the tandem SH3 domains of p47phox are well conserved.
Stabilization of the tandem SH3 domains by PBR/AIR in the autoinbitory conformation
Due to the extended structure of PBR/AIR, the buried surface area with the two SH3 domains is 3625 Å2. The interaction between the tandem SH3 domains and the short sequence of RGAPPRRSSI in PBR/AIR stabilizes the autoinhibitory conformation, where the buried surface area is 1283 Å2. Thus, the remaining PBR/AIR significantly contributes to the stabilization of the autoinhibitory conformation of p47phox.
The residues Arg301-Ser304 and Ser315-Ser320 in PBR/AIR form salt-bridges and hydrogen bonds directly and indirectly with the following residues, Leu210 and Glu211 in N-SH3; Glu241, Asp243, Glu244 and Asp261 in C-SH3, and Pro216 in the linker. Most of the interactions are mediated by water molecules and we name this interaction site, site (i) (Fig. 5A and 5B). The cluster of four arginine residues including Arg301, Arg302, Arg316 and Arg 318 (arginine cluster) in the cleft between N-SH3 and C-SH3 appears to constitute the interaction core, although the interactions mediated by a number of water molecules seem to be weak. There are several hydrogen bonds formed between PBR/AIR and N-SH3 including the following residues, His309-His312 in PBR/AIR and Arg162, Ile164, Glu211 and Pro212 in N-SH3, to which we refer as site (ii) (Fig. 5A and 5C). The interaction site between the B helix in PBR/AIR and C-SH3 is referred to as site (iii) (Fig. 5A and 5D). There are several hydrophobic interactions among the B helix, the linker and C-SH3; Val265, Ile256 (C-SH3), Tyr226 (the linker), Phe331 and Tyr324 (PBR/AIR). The B helix also forms hydrogen bonds with C-SH3, including His257, Arg267, Thr272 in C-SH3 and Gln321, Tyr324 and Ser328 in PBR/AIR.
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Discussion |
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Intramolecular interactions for maintaining the autoinhibited conformation of the tandem SH3 domains have been categorized into two groups, the direct and indirect interactions with the ligand binding surfaces of both the SH3 domains. The direct interaction of the GAPPR sequence with the tandem SH3 domains in the autoinhibited form should be loose enough to enable the ligand exchange with p22phox PRR when p47phox is activated. Meanwhile, a number of indirect interactions are utilized to stabilize the autoinhibitory conformation: the N-SH3 and C-SH3 domains interact with each other at the n-Src loops, as well as with PBR/AIR and the linker. However, each interaction seems to be loose, suggesting that the autoinhibited conformation could be maintained synergistically by a number of loose interactions. Indeed, in the absence of PBR/AIR, the tandem SH3 domains do not form a compact globular structure but exhibit an extended and flexible structure in solution based on the small-angle X-ray scattering analysis (unpublished observation). This is consistent with the evidence that the binding affinity between the tandem SH3 domains and PBR/AIR is enhanced by the presence of an additional region following the GAPPR sequence (Ago et al. 1999; Groemping et al. 2003). There are additional interactions between the linker and the SH3 domains which could play a role in maintaining the SH3 domains in the autoinhibited conformation considering that the mutations at the linker activate p47phox to bind to p22phox (Peng et al. 2003). These findings are consistent with the model in which the tandem SH3 domains in the autoinhibited form is stabilized synergistically. Although such interactions do not directly occlude the ligand binding surface of the SH3 domains, they contribute to force the tandem SH3 domains to adopt an inactive conformation. Cooperativity among the direct and indirect interactions would endow the rigorous regulation of the autoinhibited tandem SH3 domains of p47phox. This reminds us of the regulation of Src family kinases, in which the SH2 and SH3 domains bind to their intramolecular ligands to form an autoinhibited form. These interactions are released by dephosphorylation and high-affinity ligand binding so that the kinase activity is restored (Xu et al. 1997; Sicheri et al. 1997; Williams et al. 1997).
Conformational switch by phosphorylation of the specific Ser residues in PBR/AIR
The phospholyration of the specific serine residues in PBR/AIR plays a pivotal role in the activation and assembly process of the NADPH oxidase complex (Leusen et al. 1996; Babior 1999; Nauseef 1999; Segal et al. 2000). Five serine residues in PBR/AIR of p47phox, Ser303 Ser304 Ser315 Ser320 and Ser328, have been suggested to be phosphorylated by protein kinase C (PKC) or Akt (El Benna et al. 1994, 1996; Fontayne et al. 2002; Hoyal et al. 2003). The location and the environment of these phosphorylation sites in the globular module provide an insight into the activation mechanism of p47phox. The phosphorylation sites are grouped into two types, the exposed and the buried.
Ser315 and Ser320 are located on the solvent exposed region in PBR/AIR (Fig. 5A,B) and their side chains are well exposed. Ser315 and Ser320 are close to Arg316 and Arg318 in the arginine cluster which forms the potential hydrogen bond network mediated by water molecules (Fig. 5B). The introduction of negative charges at Ser320 by phosphorylation seems to be favourable because of the capping of the B helix and the electrostatic interaction with Arg318. Phosphorylation of these exposed serine residues is likely to cause conformational changes through interactions with Arg316 and Arg318, disrupting the interaction network mediated by water molecules. However, mutational analysis supports the idea that phosphorylation of Ser315 and Ser320 makes only minor or no contribution to the NADPH oxidase activation (Ago et al. 1999; Groemping et al. 2003). Phosphorylation of these residues seems to have less effect on the overall conformation of the autoinhibited tandem SH3 domains but possibly contributes to the destabilization of the autoinhibited conformation.
On the other hand, Ser303 and Ser304 in the 310 helix following the GAPPR sequence and Ser328 at the centre of the B helix are buried. The side chain of Ser303 directly forms a hydrogen bond with Glu241 and indirectly interacts with Asp243. The side chain of Ser303 is shielded by Glu241 and Arg301 (Fig. 5B insert). Phosphorylation of Ser303 seems to break the interaction network due to electrostatic repulsion with the side chain of Glu241, and disrupts the water-mediated interaction with the side chains of Glu241 and Asp243 and induces the conformational change of the side chain of Arg301, which results in a diminished interaction network in the autoinhibited conformation. The side chain of Ser304 is partially shielded by the side chain of Arg301 (Fig. 5B insert). Phosphorylation of Ser304 seems to have a less direct effect on the conformation of the autoinhibited form but does induce local conformational changes around Ser304, resulting in disruption of the interaction network formed by the side chains of Arg301 and Arg302. The side chain of Ser328 located on the B helix forms a hydrogen bond with the guanidinium group of Arg267, and is completely shielded by the Arg267 and Thr272 side chains. Phosphorylation of Ser328 could break the hydrogen bond and cause steric clash, resulting in the destabilization of the B helix (Fig. 5D).
Substitution of a combination of the serine residues: Ser303, Ser304 and Ser328, by phosphomimetic residues, glutamates or aspartates, induces the direct interaction with p22phox and leads to the activation of the NADPH oxidase (Inanami et al. 1998; Ago et al. 1999; Groemping et al. 2003). These reports are consistent with the idea that phosphorylation of these residues could destabilize the autoinhibited conformation to form the unmasked state.
At the activation step, the exposed serine residues, Ser315 and Ser320, are first phosphorylated, which seems to loosen the water-mediated interaction network to expose the buried serine residues, Ser303, Ser304 and Ser328 to the solvent. Once exposed, these serine residues could be phosphorylated. The disruption of the interaction network occurs both through charge repulsions and steric clashes, resulting in the conformational change of the tandem SH3 domains successively from the masked state to the unmasked state, thereby enabling them to bind to p22phox PRR.
The interaction between the PX domain and the tandem SH3 domains in the context of full-length p47phox
PX domains were identified as the phosphatidylinositol phosphate binding domains (reviewed in Sato et al. 2001). The PX domain of p47phox assists to tether p47phox to the membrane to activate the NADPH oxidase. In the resting state, however, its binding was reported to be suppressed by the intra-molecular interaction in the context of the full-length p47phox (Karathanassis et al. 2002; Ago et al. 2003). This is consistent with the experimental result that the isolated C-SH3 domain binds to the PXXP motif of the isolated PX domain, thus enabling the PX domain inaccessible to the binding to the phosphatidylinositol phosphates (Hiroaki et al. 2001).
However, the present crystal structure demonstrated that the GAPPR sequence in PBR/AIR occupied the conserved PRR binding surface of both N-SH3 and C-SH3 so that C-SH3 is not available for the interaction with the PXXP motif on the PX domain. The C-SH3 domain in the autoinhibited form might interact with the PX domain using other surface but not the conserved PRR binding surface. The binding ability of the PX domain to the phosphatidylinositol phosphates in full-length p47phox is restored once the autoinhibitory conformation is released by the phosphorylation (Karathanassis et al. 2002; Ago et al. 2003). We need further structural studies in the context of full-length p47phox to clarify the inhibition mechanism of the PX domain inaccessible to the phosphatidylinositol phosphates in the resting state.
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Experimental procedures |
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The sample preparation and crystallization condition of p47phox(151-340) were previously described (Yuzawa et al. 2003). The native data were collected at BL44XU of SPring-8 in Hyogo Japan using a DIP 6040 detector with a crystal-to-detector distance of 100 mm at 100 K. For phase determination by the multiwavelength anomalous diffraction (MAD) method (Hendrickson et al. 1990), we prepared a selenomethionine (SeMet)-substituted protein. The SeMet derivative was expressed in Escherichia coli B834(DE3) using an amino acid medium (LeMaster & Richards 1985) containing SeMet instead of methionine and crystallized under conditions nearly identical to those for the native protein. The SeMet-substituted crystal was smaller than the native crystal. A set of MAD data using three wavelengths was collected at BL41XU of SPring-8 in Hyogo, Japan using a mar CCD 165 detector with a crystal-to-detector distance of 150 mm at 100 K from a single crystal for SeMet-substituted p47phox(151-340). The MAD and native data were processed and scaled using the HKL2000 suite of program (Otwinowski & Minor 1997).
Structure solution and refinement
All of the programs used for heavy-atom searches, phasing and refinement were those attached to the Crystallography and NMR System version 1.1 (CNS) package (Brünger et al. 1998). Only three (Se175, Se198 and Se279) out of four potential selenium sites in the asymmetric unit were found by using an automated Patterson heavy atom search method (Grosse-Kunstleve & Brünger 1999) and peak anomalous wavelength data. The experimental phase was calculated and refined from a combination of MAD and native data using selenium coordinates. The phases were further improved by solvent flipping (Abrahams & Leslie 1996). The resulting electron-density maps were of interpretable quality at 3.5 Å resolution and allowed the construction of a molecular model of p47phox(151-340). The model was built manually using the Turbo-Frodo program (Cambillau & Roussel 1997) in the experimental electron density maps. An initial refinement was performed by the torsion angle molecular dynamic simulated annealing method and bulk-solvent correction against the maximum-likelihood amplitude target. For each refinement cycle, the model was rebuilt manually. Refinement was performed by energy minimization, individual isotropic B-factor refinement, and bulk-solvent correction against the maximum-likelihood amplitude target. Results of refinement trails were monitored with a random selection of 10% of the data used for cross-validation (Brünger 1992). Subsequently, the refined model of the SeMet-substituted protein was used to solve the structures of the native protein.
Structure determination
After refinements, the final crystallographic model includes 1394 atoms (170 residues) plus 154 oxygen atoms from H2O. Residues that are not visible in the maps include the first nine N-terminal residues (148–156) containing three residues derived from a cloning artifact, as well as the last four C-terminal residues (337–340) of the native protein. The three residues, 170–172 in the RT-loop of the N-terminal SH3 domain (N-SH3) could not be modelled due to poor electron density. In residues 167–176, B-factors of all atoms are relatively high (> 40 Å2), indicating extensive flexibility of the polypeptide chain. In addition, some solvent-exposed side chain atoms of the residues Ile157, Glu168, Lys169, Glu174, Lys201, Asp269, Asp270, Arg314, Arg335 and Arg336 could not be modelled into the electron-density map and were therefore omitted from the final model. The structure of p47phox(151-340) has a R-value of 21.3% and a free R-value of 23.6% at 1.82 Å resolution against the native data set (Table 1). The structural quality assessed by the PROCHECK program (Laskowski et al. 1993) indicates that 94.2% of the residues are in the most favourable region except Gly and Pro, while none of the residues are in the disallowed region of the Ramachandran plot. All of the data collection, phasing and refinement statistics are summarized in Table 1. The coordinates of p47phox(151-340) have been deposited in the Protein Data Bank, with accession code 1UEC [PDB] . All structure figures were prepared using PyMOL (DeLano 2001).
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Acknowledgements |
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The atomic coordinates and structure factors (PDB code, 1UEC [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics. Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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Footnotes |
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* Correspondence: E-mail: finagaki{at}pharm.hokudai.ac.jp
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References |
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Received: 25 December 2003
Accepted: 3 February 2004
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positions. (B, C) Stick model representation of interaction network of the linker with both SH3 domains. The N-terminal region of the linker mainly interacts with N-SH3 (B), and the C-terminal region of the linker interacts with C-SH3 (C), which we refer to A and B sites, respectively.