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Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47phox, regulating NADPH oxidase activity
Computational Biology and Chemistry 30 (2006) 303–312
Case study
Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47phox, regulating NADPH oxidase activity Yoko Watanabe a , Hideyuki Tsuboi a , Michihisa Koyama a , Momoji Kubo a,b , Carlos A. Del Carpio a , Ewa Broclawik c , Eiichiro Ichiishi c , Masahiro Kohno c , Akira Miyamoto a,c,∗ a
Department of Applied Chemistry, Graduate School of Engineering, Tohoku University, 6-6-11-1302 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan b PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan c New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan Received 16 June 2005; received in revised form 10 April 2006; accepted 24 April 2006
Abstract The phagocyte NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of superoxides. Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by the absence of certain components of the NADPH oxidase. Key to the activation of the NADPH oxidase is the cytoplasmic subunit p47phox , which includes the tandem SH3 domains (N-SH3 and C-SH3). In active phagocytes, p47phox forms a stable complex with the cytoplasmic region of membrane subunit p22phox that forms a left-handed polyproline type-II (PPII) helix conformation. In this report, we have analyzed the conformational changes of p47phox –p22phox complexes of wild-type and three mutants, which have been detected in CGD patients, using molecular dynamics simulations. We have found that in the wild-type, two basal planes of PPII prism in cytoplasmic region of p22phox interacted with N-SH3 and C-SH3. In contrast, in the modeled mutants, the residue at the ape of PPII helix, which interacts simultaneously with both of the tandem SH3 domains in the wild-type, moved toward C-SH3. Furthermore, interaction energies of the cytoplasmic region of p22phox with C-SH3 tend to decrease in these mutants. All these findings led us to conclude that interactions between N-SH3 of p47phox and PPII helix, which is formed by cytoplasmic region of p22phox , may play a significant role in the activation of the NADPH oxidase. © 2006 Elsevier Ltd. All rights reserved. Keywords: Molecular dynamics; NADPH oxidase; SH3 domain; PPII helix; Ligand recognition
The phagocyte NADPH oxidase is found primarily in neutrophils complex and plays a crucial role in the innate immune system that defends the host against bacterial, fungal, and viral pathogens. NADPH oxidase plays an important role in the defense system of human body by catalyzing the NADPH oxidation (Eq. (1)), which leads to the production of superoxide from oxygen and NADPH (Ago et al., 1999; Thrasher et al., 1994) NADPH + 2O2 → NADP+ + H+ + 2O2 −
∗
Corresponding author. Tel.: +81 22 795 7233; fax: +81 22 795 7235. E-mail addresses: [email protected] (A. Miyamoto), [email protected] (H. Tsuboi), [email protected] (M. Koyama), [email protected] (M. Kubo), [email protected] (C.A. Del Carpio), [email protected] (E. Broclawik), [email protected] (E. Ichiishi), [email protected] (M. Kohno). 1476-9271/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.compbiolchem.2006.04.004
(1)
Superoxides are converted, in turn, by means of enzymatic and non-enzymatic pathways to more potent oxidizing agents (Miller and Britigan, 1995). Neutrophils localize NADPH oxidases into the phagolysosomal membrane surrounding engulfed microbes which are killed by the oxidizing agents generated by NADPH oxidases. Inappropriate activation of this enzyme leads to the overproduction of superoxide, resulting in inflammatory responses and a number of disease states including chronic inflammation (Benard et al., 1999). Therefore, controlling superoxide production requires a detailed understanding of the activation mechanism of NADPH oxidase. In the resting state, the NADPH oxidase subunits are localized separately at the membrane and cytoplasm. The catalytic core of NADPH oxidase is the integral membrane protein cytochrome b558 that consists of gp91phox and p22phox subunits (Huang et al., 1995). However, cytochrome b558 is inactive in the absence of at least three cytoplasmic subunits p47phox , p67phox , and the small GTPase Rac that translocate to the membrane and
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Fig. 1. Domain organization of NADPH oxidase subunits (A) p47phox and (B) p22phox . PX, SH3, P, and T represent phox homology domain, Src homology domain 3, proline-rich region, and transmembrane domain, respectively.
bind cytochrome b558 during phagocytosis (DeLeo and Quinn, 1996). Key to this translocation process is p47phox , which consists of a PX domain, N-terminal SH3 domain (N-SH3), C-terminal SH3 domain (C-SH3), a polybasic region, and a proline-rich region (Fig. 1(A)). The tandem SH3 domains bind to the C-terminal cytoplasmic proline-rich region of p22phox (Fig. 1(B)), and this interaction is essential for NADPH oxidase activity (Sumimoto et al., 1996). In the inactive state, the binding surface of the tandem SH3 domains is masked through an intra-molecular interaction with polybasic region, resulting in the auto-inhibited form (Ago et al., 1999; Huang and Kleinberg, 1999; Sumimoto et al., 1994; Leto et al., 1994). In response to phagocyte activation, several serine residues located in the polybasic region of p47phox are phosphorylated, leading to changes in the conformation of p47phox and exposure of the binding surface of the tandem SH3 domains (Groemping et al., 2003; Ago et al., 2003). Chronic granulomatous disease (CGD) is caused by mutations in genes encoding the four PHOX subunits of NADPH oxidase (Casimir et al., 1992). Mutations in these genes cause a significant decrease in production of superoxide, and accordingly, CGD patients repeat life-threatening infections. Specifically, the gene for gp91phox , which is on the X chromosome, accounts for approximately 60% of all CGD cases. The rest of PHOX proteins, p22phox , p47phox , and p67phox , are autosomal and account for the remainder cases of CGD. Studies have shown that defects in p67phox and p22phox each account for only 5% of CGD patients (Roos et al., 1996). The remaining 30% of patients have defects in p47phox , indicating the importance of p47phox to NADPH oxidase activity. Recently, Groemping et al. (2003) determined crystal structure of the active state of p47phox , which forms a complex with the C-terminal cytoplasmic proline-rich region of p22phox , by multi-wavelength anomalous diffraction. Gly 192, 262 of p47phox and Pro 156 of p22phox are involved in this complex, and are important residues whose mutations cause CGD. Groemping et al. (2003) also measured the binding affinity of this complex in order to investigate the influences of mutation sites in p47phox –p22phox complex. Mutations in the SH3 domain of p47phox reduced the affinity for the p22phox . However, the influences of mutants on the structure of complex and on the inter-molecular interaction, which cause such reduction of binding affinity, remain to be elucidated. While computational chemistry approaches are effective to derive information at the atomic level, which cannot be easily obtained from experimental approach, to our knowledge, no computational chemistry study has been performed on this system so far.
In the present study we attempt to gain insights into the submolecular mechanisms triggering the production of superoxides using available computational chemical methods. To achieve this goal, we focus concretely on conformational changes that are induced by mutations in the involved proteins that are known to be the cause of CGD in humans. Three mutants, detected in CGD patients, are mainly studied in order to elucidate the factors that determine the superoxide-generating ability of NADPH oxidase. We simulated the dynamics of p47phox –p22phox complexes of the wild-type and three mutants, and investigated the plausible changes in the inter-molecular interactions between SH3 domains of p47phox and the cytoplasmic region of p22phox . The results derived by these simulations deem an important controlling role in the activity of NADPH oxidase to the interaction between N-SH3 of p47phox and the PPII helix region of p22phox . Consequently, NADPH oxidase may not be able to produce super oxides when p47phox does not form a complex with p22phox . Furthermore, lack of interaction between N-SH3 regions corresponding to p47phox and p22phox , which is induced by change of the positional relationship between the tandem SH3 domains and the PPII helix region of p22phox , seems to also affect the superoxide-generating ability of the system. 1. Methods In the present study all the calculations we used the OPLSAA force field (Jorgensen et al., 1996) for intra- and intermolecular interactions and the surface generalized Born (SGB) model (Zhang et al., 2001; Ghosh et al., 1998) for the solvent electrostatic effects as implemented in the IMPACT program (Schr¨odinger, Inc.). 1.1. OPLS-AA force field The OPLS-AA force field is known as a force field that well reproduces liquid state properties of molecules. This force field uses empirical experimental data from the liquid state and quantum mechanical calculations to determine parameters for intra-molecular bond, angle, and torsion motions to set the constituent parameters. The intra-molecular interaction is given as, Vintra = Kr (r − req )2 + Kθ (θ − θeq )2 + Vtorsion (2) bonds
angles
where Kr and Kθ represent the force constants, and r and θ are bond lengths and angles, respectively. Suffix eq means equilibrium value. Torsional interaction is assessed as given in, Vtorsion =
Vi
1
i
+
2
[1 + cos(ϕ)] +
V3i [1 + cos(3ϕ)] 2
V2i [1 − cos(2ϕ)] 2 (3)
where the torsional interaction is summed over all the dihedral angles ϕi , and V1 –V3 are the Fourier coefficients. The nonbonded interaction is given as a van der Waals term together
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with an electrostatic term, σij12 σij6 qi qj 4εij − 6 + Vinter = Rij R12 Rij ij i>j
D=
1.2. Surface generalized Born model Recently, extensive efforts have been directed to the development of implicit solvation models for simulations of biomolecular systems that are physically reasonable and simultaneously calculation cost-effective. In SGB models, which are based on approximations of the Poisson–Boltzmann equation, the solute is described in atomic detail, while the solvent is replaced by a dielectric continuum. The Born equation gives the electrostatic free energy of transferring a spherical charged ion from a medium of dielectric constant εi to a medium of dielectric constant εo (Born, 1920). For a real molecule with an arbitrary-shaped cavity formed by the molecular surface, the electrostatic free energy of solvation is given as follows n n qi qj 1 1 1 − GGB = − 2 εi εo rij2 + α2ij e−D i=1 j=1 1 2
1 − 2
Gpair
1 =− 2
Gsingle
1 =− 2
1 1 − εi εo 1 1 − εi εo
1 1 − εi εo
n n
qi qj 2 2 −D i=1 j=1 j=i rij + αij e
n i=1
qi2 = Gpair + Gsingle αi
n n
1 1 − εi εo
i=1 j=1 j=i
n i=1
qi2 αi
rij2
(11)
(2αij )2
(4)
where qi and qj represent permanent Coulomb charges, σ ij and εij are the Lennard–Jones parameters, Rij is the inter-atomic distance. The same expression is used for intra-molecular nonbonded interactions between all pairs of atoms separated by three or more bonds. The (1,4)-interactions are scaled by a factor of 1/2. The non-bonded parameters ε and σ for each atom-pair is constructed from the atomic values by the geometric mean combination rule, √ (5) εij = εi εj √ σij = σi σj (6)
=−
305
qi qj rij2 + α2ij e−D
(7)
(8)
(9)
Eq. (9) is called the generalized Born equation (Still et al., 1990), where q and α are charge and the radius of the atom, respectively, and rij is the distance between atoms i and j. Here, αij and D are expressed as Eqs. (10) and (11), respectively, √ (10) αij = αi αj
Gsingle is the self-energy, which has exactly the same form as the Born equation, and Gpair is the pair energy. In SGB model, the effective Born radius αi can be obtained from Eq. (9) and the following equation, ∆Gsin gle
1 =− 8π
1 1 − εi εo
i
× (R − ri ) n(R) d 2 R
s
qi2 |R − ri |4 (12)
Here, ri represents the coordinates of atom i, and n(R) represents the vector of integration over the surface of the molecule (R). Then the total electrostatic free energy of solvation can be obtained by the generalized Born equation (Eq. (7)) with the effective Born radius for every atom of the solute molecule. Further details of OPLS-AA and SGB models are available in (Jorgensen et al., 1996) and (Zhang et al., 2001; Ghosh et al., 1998), respectively. 1.3. Models and conditions X-ray crystal structure of p47phox complexed with p22phox was obtained from the Protein Data Bank (PDB code: 1OV3 (Groemping et al., 2003)). The initial structure for the wildtype was obtained by refining this X-ray crystal structure using molecular mechanics energy minimization including the reported crystal water molecules. The water molecules were then removed from the finally obtained structure. We determined the initial structure of three mutants, G192S, G262S, and P156Q, based on the initial structure of the wild-type using the rotamer library of Swiss-PDB viewer (Guex and Peitsch, 1997). In mutants G192S and G262S, Gly 192 and Gly 262 of p47phox are substituted to Ser, and in P156Q, Pro 156 of p22phox is substituted to Gln. Additional minimization around the mutation site was performed for these modeled mutants. In molecular dynamics simulations, the velocity form of the Verlet algorithm (Verlet, 1967) was used with a time step of 2.0 fs to integrate the equations of the motions. 500,000 steps MD calculations were performed at 310 K for each structure. The atoms located in the N- and C-terminal residues of p47phox , and in the N-terminal (cytosolic side) residue of p22phox were treated as “buffered”. Buffered atoms are allowed to move, subject to harmonic penalty-function restraints that tether them to their initial positions. 1.4. Visualization and analysis Molecular graphics images were created with UCSF Chimera code (Pettersen et al., 2004), and interactions between protein and ligand were analyzed by schematic representation using the LIGPLOT program (Wallace et al., 1995).
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2. Results and discussion 2.1. Validation of the initial structure and molecular dynamics simulations As mentioned above, we first performed structure optimization of the protein structure with crystal water molecules to determine the initial structure, because these molecules are important for protein stability. Fig. 2(A) shows the root mean square deviations (RMSD’s) between corresponding residues of the X-ray crystal structure and the obtained initial structure. RMSD’s values are small for the ␣-helix and -strand regions of the protein, which are indicated by arrows in Fig. 2(C), while RMSD’s values are large for the loop region. This is a sign that the initial structure was adequately generated from the X-ray crystal structure of p47phox –p22phox complex. In the successive molecular dynamics simulations, the crystal water molecules were removed and the SGB model was applied in order to evaluate the solvent effect. The adequacy of this approximation was confirmed by a meticulous estimation of the structural changes arisen on each model. Similarly as for the initial structure, RMSD values for corresponding residues in the initial and final structures are large in the loop region while the RMSD values are small in the ␣-helix and -strand regions. Moreover, none significant structural change in the wild-type (Fig. 2(B)) was observed, confirming thus the adequacy of the series of these simulations. 2.2. A novel mode of SH3–ligand interaction of p47phox In the active and inactive phagocyte, the SH3 domains of p47phox form a complex with the cytoplasmic region of p22phox and the polybasic region of p47phox , respectively. Both of these two ligands form a PPII helix conformation. Analysis of the initial structure, computed here by energy minimization, shows an agreement with this observation since the cytoplasmic region of p22phox interacts with both of N-SH3 and C-SH3 in the active phagocyte (Fig. 3). The binding of the tandem SH3 domains of p47phox and cytoplasmic region of p22phox is illustrated in
Fig. 2. Root mean square deviation (RMSD) of the initial and final structures of p47phox –p22phox complex against residue number. (A) RMSD of the initial structure from X-ray crystal structure. (B) RMSD of each final structure from the initial structure. Residues located in N-SH3 and C-SH3 of the final structure were superimposed on that of the initial structure, and then RMSD was calculated. (C) Secondary structure of p47phox . The arrows indicate ␣-helices and -strands.
Fig. 3. Inter-molecular interaction between the tandem SH3 domains of p47phox and cytoplasmic region of p22phox . The residues of cytoplasmic region of p22phox are shown in purple. The residues of N-SH3 and C-SH3 of p47phox were drawn in left side and right side of p22phox , respectively. Hydrogen bonds and hydrophobic interactions are indicated black dashed lines and green semi-circles, respectively.
Fig. 4(A). Residues 154–158 in p22phox form a PPII helix conformation that is characterized by a triangular prism-like structure. The basal plane formed by residues Asn 154, Pro 156 and Pro 157 interact with N-SH3, while the basal plane formed by Pro 155, Pro 156, and Arg 158 with C-SH3. Pro 156, located at the apex of the prism and formed by those basal planes, interacts with N-SH3 and C-SH3, simultaneously. Since this synergetic interaction, stabilizes the complex (as shown by our calculations of the initial structure), destabilization of the same can be assumed to be directly dependant to the formation or not of this particular spatial arrangement or conformation of the amino acids in the interacting subunits. And, by extension, this conformation can be thought to play an important role in the activation of p47phox . Our assumption is further supported by the fact that this interaction is radically different in other SH3 domain/target complexes. Moreover, molecular recognition between SH3 domain and PPII helix has been elucidated by X-ray crystallography and NMR, and is well characterized (Mayer, 2001; Kuriyan and Cowburn, 1997). These reported experimental observations stand for a PPII helix that takes on a structure analogical to a triangular prism, where residues at the single basal plane bind to a single SH3 domain, while those at the apex are exposed. In sharp contrast to these observations, Groemping et al. (2003) and Yuzawa et al. (2004) have found a different
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307
Fig. 4. Binding of the tandem SH3 domains of p47phox and its ligands that are forming PPII helix conformation. (A) Tandem SH3 domains of p47phox and cytoplasmic region of p22phox (residues 154–158) in the initial structure determined by structure optimization. (B) X-ray crystal structure of the tandem SH3 domains of p47phox and polybasic region of p47phox (residues 297–301) (Yuzawa et al., 2003).
mode of SH3–ligand interaction in the X-ray crystal structure of auto-inhibited p47phox , where the two basal planes of the PPII prism interact with N-SH3 and C-SH3 in auto-inhibited p47phox as shown in Fig. 4(B). This indicates that the polybasic region of p47phox does not bind tightly to each SH3 domain enabling in this way the ligand exchange with the cytoplasmic region of p22phox when p47phox is activated. On the contrary, auto-inhibited p47phox is stabilized by the synergic interaction between the tandem SH3 domains and polybasic region of p47phox (Groemping et al., 2003; Yuzawa et al., 2004). We found that the mode of inter-molecular interaction between the tandem SH3 domains of p47phox and the cytoplasmic region of p22phox observed in our simulations is the same as that of the intra-molecular interaction in p47phox whose X-ray structure has been reported in the literature (Groemping et al., 2003; Yuzawa et al., 2004). This leads to the conclusion that the same mode of interaction between the SH3 domain and its ligand is required for both of the active and inactive states of NADPH oxidase. 2.3. The role of the mutation sites in the activation of NADPH oxidase
between the two SH3 domains. On the contrary, the distance between Ser 262 and Ile 159 remained almost intact as well as the distance between Gly 262 and Ile 159 (Fig. 6(C)). In the initial structure of the wild-type, the sidechains of Trp 193 and Trp 263 form hydrogen bonds with the backbone carbonyl oxygen atoms of Asn 154 and Pro 156 of p22phox , connecting the cytoplasmic region of p22phox with N-SH3 and C-SH3 of p47phox , respectively (Fig. 5). While the mutation of Gly 262 to serine has almost no perceptible effect on the atomto-atom distance between Trp 263 and Pro 156 (Fig. 6(D)), the mutation of Gly 192 to serine increases these distances between Trp 193 and Asn 154 (Fig. 6(B)). In contrast, mutation of Pro 156 to glutamine affects the atom-to-atom distance between Trp 263 and Gln 156 (Fig. 6(D)). Although both, Gly 192 and 262 are residues in n-Src loop of p47phox , only structural changes in G262S are less severe than that in G192S. This result is in fair agreement with isothermal titration calorimetry measurements carried out by Groemping et al. (2003), which report that the affinity of p47phox with p22phox is reduced both in G192S and G262S, and the effect is more significant in G192S than in G262S. From our simulations, it can be inferred that the increased instability of G262S as compared with G192S is the result of the lack of intra-molecular interaction between the two
Since clinical data show the presence of particular mutants of p47phox as well as p22phox in patients with CGD, we carried out a further analysis of the plausible interaction patterns of the mutants and compared them with that in the wild type complex. Mutated structures of p47phox –p22phox complex were obtained using molecular dynamics simulations. In order to elucidate the structural changes caused by the mutations, we analyzed the hydrogen bonds appearing and disappearing between the mutated sites and the residues around them, as compared to those in the wild type complex. In the initial structure of the wild-type, which was determined by structure optimization, the amide nitrogen atom of Gly 192 and Gly 262 form hydrogen bond with the backbone carbonyl oxygen atom of Ser 189 and Ile 259, respectively (Fig. 5). These hydrogen bonds stabilize the n-Src loop in N-SH3 and C-SH3, which mediates the interaction between the two SH3 domains. When Gly 192 is mutated to serine, the distance between Ser 192 and Ser 189 increases (Fig. 6(A)), indicating that the mutation of Gly 192 to serine disrupts the n-Src loop of N-SH3 and consequently the interaction
Fig. 5. Hydrogen bonds formed by the mutation site or the residues around it. Hydrogen bonds are indicated with purple lines. The residues located in N-SH3, C-SH3 and p22phox are shown in light blue, light green, and pink.
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Fig. 6. The changes in atom–atom distance shown in Fig. 5. Atom–atom distances between (A) the amide nitrogen atom of Gly (Ser in G192S) 192 and the backbone carbonyl oxygen atom of Ser 189; (B) the sidechain of Trp 193 and the backbone carbonyl oxygen atom of Asn 154; (C) the amide nitrogen atom of Gly (Ser in G262S) 262 and the backbone carbonyl oxygen atom of Ile 259; (D) the sidechain of Trp 263 and the backbone carbonyl oxygen atom of Pro (Gln in P156Q) 156.
SH3 domains due to the disruption of the n-Src loop in N-SH3. Additionally, Pro 156 stabilizes the inter-molecular interaction between C-SH3 domain of p47phox and the cytoplasmic region of p22phox and is important to form p47phox –p22phox complex. 2.4. Inter-molecular interaction between p47phox and p22phox Mutation at Gly 192, Gly 262 of p47phox and Pro 156 of p22phox leads to a difference in the interaction energy of the cytoplasmic region of p22phox with the SH3 domains of p47phox . This difference was estimated computing the interaction energies for
the complexes using the following equation: Einteraction = Ecomplex − (Ereceptor + Eligand )
(13)
where Ecomplex is the total energy of the p47phox –p22phox complex, and Ereceptor and Eligand are respectively those of the receptors (tandem SH3 domains, N-SH3, and C-SH3) and ligands (SH3-binding region and PPII helix region). Complexes of N-SH3-p22phox and C-SH3-p22phox were minimized around p22phox before calculating the interaction energy. Fig. 7 shows the changes in interaction energy for the SH3-binding region of p22phox with the SH3 domains, and Table 1 shows the interaction energy for the PPII helix region of p22phox with the SH3
Table 1 Interaction energy of PPII helix region of p22phox with the SH3 domains of p47phox in the initial structures determined by structure optimization Model Wild-type G192S G262S P156Q Wild-type G192S G262S P156Q Wild-type G192S G262S P156Q
Receptor
Tandem SH3
N-SH3
C-SH3
Ligand
Interaction energy (kcal/mol)
PPII helix region of p22phox
−74.224 −56.911 −74.561 −69.111
PPII helix region of
p22phox
−23.944 −18.038 −23.831 −27.648
PPII helix region of
p22phox
−51.034 −50.768 −58.264 −42.015
Tandem SH3 domains, N-SH3, and C-SH3 correspond to residues 159–283, 159–213, and 229–283 of p47phox . SH3-binding region and PPII helix region correspond to residues 150–160 and 154–158 of p22phox .
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309
Fig. 7. Interaction energy of SH3-binding region of p22phox with the SH3 domains of p47phox during molecular dynamics simulation. (A) Wild-type, (B) G192S, (C) G262S, and (D) P156Q. Interaction energy of SH3-binding region with the tandem SH3, N-SH3 and C-SH3 are shown in gray, black, and white, respectively.
domains in the initial structure, respectively. While the interaction energy of the SH3-binding region with N-SH3 increases proportionally to the simulation time in the wild-type complex, interaction energies decrease in three of the mutants. On the contrary, interaction energies of SH3-binding region with the tandem SH3 domains and C-SH3 do not change significantly during these simulations. This can be considered an indication that the lack of interaction between N-SH3 of p47phox and the cytoplasmic region of p22phox induces inactivation of NADPH oxidase. In initial structures of all the investigated cases, which were determined by structure optimization, interaction energies of SH3-binding region with N-SH3 are almost the same as those with C-SH3 (Fig. 7). On the contrary, interaction energies of PPII helix region with C-SH3 have apparently less value than those with N-SH3 (Table 1), indicating stronger interaction. Therefore, we can conclude from our calculations that the cytoplasmic region of p22phox forms a stable complex with both of the tandem SH3 domains, and that the PPII helix region interacts mainly with C-SH3. Additionally this PPII helix conformation contributes significantly to the interaction between p47phox and p22phox , and can be deemed one of the important factors in the determination of the active/inactive state of NADPH oxidase.
mic region of p22phox . To analyze this hypothesis we performed further molecular dynamic simulations of this protein complex. Since molecular dynamics simulations produce large numbers of conformations for the wild-type and mutated p47phox –p22phox complexes, to study the structural changes in the cytoplasmic region of p22phox . We focused on the low energy conformations of these complexes. The total energy curves for the molecular dynamics simulations have local minimum values at several points. As shown in Fig. 8, the total energy of the wild-type has local minimum values at 520, 870, 910, and 950 ps, G192S at 740 and 870 ps, G262S at 400, 770, and 810 ps, P156Q at 250 and 300 ps. We considered that the structures at these time steps exist as stable conformations and thus analyzed
2.5. Structural changes of the cytoplasmic region of p22phox The observation of the decrement in interaction energy between N-SH3 of p47phox and the cytoplasmic region of p22phox in three of the analyzed mutants, may also be attributed to conformational change of the PPII helix region of the cytoplas-
Fig. 8. Total energy changes, including SGB energy, during the molecular dynamics simulations.
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them in detail. However, the relationship among all the generated structures cannot be neglected if a complete analysis of the problem is targeted, as is the case of the present work. Therefore, we performed a cluster analysis to filter minor conformations using the XCluster software (Shenkin and McDonald, 1994) which is a tool for observing and analyzing the molecular conformations based on molecular similarity criteria. Our analysis revealed that structures, which energy values similar to local minima structures are observed with high frequency during molecular dynamics simulations. This means that these structures exhibit stable conformations and are not just mere minor conformations. In order to infer the cause of decrease of interaction energy between N-SH3 of p47phox and the cytoplasmic region of p22phox in three mutants using computational instances alone, we analyzed the conformational change of the PPII helix region in the cytoplasmic region of p22phox (residues 154–158) using these low energy conformations. Before the conformational analyses we performed an energy minimization process around p22phox . Fig. 9 shows the Ramachandran plot for residues 154–158 in p22phox of low energy structures. Plots for the wildtype, G192S, and G262S are around the plot for typical PPII helix (Fig. 9(A)–(C)), meaning that these residues maintain the original PPII helix conformation during the molecular dynamics simulations. However, a difference in the positional relationship between the tandem SH3 domains and PPII helix region in the wild-type, G192S, and G262S mutants can be observed. Fig. 10 shows the structure of the tandem SH3 domains and PPII helix region after molecular dynamics simulation. In the wild-type,
Fig. 9. Ramachandran plots for PPII helix region of cytoplasmic region of p22phox (residues 154–158) during the molecular dynamics simulations. (A) Wild-type, (B) G192S, (C) G262S, and (D) P156Q. Phi and psi designate the rotation angle around the N–C␣ and C␣ –C bonds in polypeptide backbone, respectively. The blue solid lines and light blue regions indicate the range of allowed and core regions (Morris et al., 1992), respectively. The black solid line indicates the plot of typical PPII helix.
Fig. 10. The tandem SH3 domains of p47phox and cytoplasmic region of p22phox during molecular dynamics simulation. (A) Wild-type at 950 ps, (B) G192S at 870 ps, (C) G262S at 810 ps, and (D) P156Q at 300 ps. PPII helix region of p22phox (residues 154–158) and the mutation site are shown in pink and yellow, respectively.
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two basal planes of the PPII prism interact with both of the tandem SH3 domains (Fig. 10(A)) in the same manner as the initial structure determined by structure optimization. In contrast, the backbone of Pro 156 that interacted with both N-SH3 and CSH3 in the initial structure interacts only with C-SH3 in G192S and G262S (Fig. 10(B) and (C)). In P156Q, Ramachandran plot deviates from typical PPII helix, meaning that residues 154–158 cannot maintain PPII helix conformation during the molecular dynamics simulation (Fig. 9(D)). Moreover, P156Q shows no local minimum value after 300 ps. Therefore, we can hypothesize that P156Q cannot form a stable p47phox –p22phox complex, because the PPII helix conformation in cytoplasmic region of p22phox is disrupted. 3. Conclusions In the present study we used computational methodologies to investigate the most important structural factors of the system p47phox –p22phox that may be involved in the activation of NADPH and its superoxide-generating ability. Our study reveals that the SH3 domain may play an important role as the key mediator of interactions that regulate the formation of NADPH oxidase complex and enzyme activity. The calculations show that the cytoplasmic region of p22phox interacts synergetically with the tandem SH3 domains of p47phox , leading us to hypothesize that this results in the activation of NADPH oxidase. Our simulation results of p47phox –p22phox complexes for wild-type and mutants show the characteristics of this intermolecular interaction. Our simulation results also reveal that the PPII helix conformation of the cytoplasmic region of p22phox plays an indispensable role in the formation of p47phox –p22phox complex. In spite of the synergetic interaction in p47phox –p22phox complex, the PPII helix region interacted mainly with C-SH3. In all of the investigated mutants, that are not able to produce superoxide, interaction energies of SH3binding region with N-SH3 decrease. However, there is a difference in the inactivation mechanism of the investigated mutants. The cytoplasmic region of p22phox maintains PPII helix conformation, and synergic interaction between PPII helix region and tandem SH3 domains of p47phox is disrupted in G192S and G262S. On the contrary, the cytoplasmic region of p22phox cannot maintain PPII helix conformation in P156Q. The information obtained from our simulations and the subsequent analysis illustrates the inter-molecular interaction mode at the sub-molecular level between p47phox and p22phox that may be intimately related to the causes of inactivation of NADPH oxidase, and can be deemed of great importance for further advances in the medical treatment of CGD and chronic inflammation. References Ago, T., Nunoi, H., Ito, T., Sumimoto, H., 1999. Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47phox . J. Biol. Chem. 274, 33644–33653. Ago, T., Kuribayashi, F., Hiroaki, H., Takeya, R., Ito, T., Kohda, D., Sumimoto, H., 2003. Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides. Leading to phago-
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cyte NADPH oxidase activation. Proc. Natl. Acad. Sci. U.S.A. 100, 4474–4479. Benard, V., Bokoch, G.M., Diebold, B.A., 1999. Potential drug targets: small GTPases that regulate leukocyte function. Trends Pharmacol. Sci. 20, 365–370, doi:10.1016/S0165-6147(99)01367-X. Born, M., 1920. Volumen und hydratationswarme der ionen. Z. Phys. 1, 45–48. Casimir, C., Chetty, M., Bohler, M.C., Garcia, R., Fischer, A., Griscelli, C., Johnson, B., Segal, A.W., 1992. Identification of the defective NADPHoxidase component in chronic granulomatous disease: a study of 57 European families. Eur. J. Clin. Invest. 22, 403–406. DeLeo, F.R., Quinn, M.T., 1996. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J. Leukocyte Biol. 60, 677–691. Ghosh, A., Rapp, C.S., Friesner, R.A., 1998. Generalized Born model based on a surface integral formulation. J. Phys. Chem. B 102, 10983–10990, doi:10.1021/jp982533o. Groemping, Y., Lapouge, K., Smerdon, S.J., Rittinger, K., 2003. Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113, 343–355, doi:10.1016/S0092-8674(03)00314-3. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723, doi:10.1002/elps.1150181505. Huang, J., Kleinberg, M.E., 1999. Activation of the phagocyte NADPH oxidase protein p47phox . J. Biol. Chem. 274, 19731–19737. Huang, J., Hitt, N., Kleinberg, M.E., 1995. Stoichiometry of p22phox and gp91-phox in phagocyte cytochrome b558 . Biochemistry 34, 16753–16757. Jorgensen, W.L., Maxwell, D.S., Tirado-Rives, J., 1996. Development and testing of the OPLS all-atom force field on conformation energetics and properties of organic liquids. J. Am. Chem. Soc. 118, 11225–11236, doi:10.1021/ja9621760. Kuriyan, J., Cowburn, J., 1997. Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288, doi:10.1146/annurev.biophys.26.1.259. Leto, T.L., Adams, A.G., de Mendez, I., 1994. Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets. Proc. Natl. Acad. Sci. U.S.A. 91, 10650–10654. Mayer, B.J., 2001. SH3 domains: complexity in moderation. J. Cell Sci. 114, 1253–1263. Miller, R.A., Britigan, B.E., 1995. The formation and biologic significance of phagocyte-derived oxidants. J. Invest. Med. 43, 39–49. Morris, A.L., MacArthur, M.W., Hutchinson, E.G., Thornton, J.M., 1992. Assessing the accuracy of protein structural coordinates. Proteins: Struct. Funct. Genet. 12, 345–364. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., 2004. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612, doi:10.1002/jcc.20084. Roos, D., de Boer, M., Kuribayashi, F., Meischl, C., Weening, R.S., Segal, A.W., Ahlin, A., Nemet, K., Hossle, J.P., Bernatowska-Matuszkiewicz, E., Middleton-Price, H., 1996. Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease. Blood 87, 1663– 1681. Shenkin, P.S., McDonald, D.Q., 1994. Cluster analysis of molecular conformations. J. Comput. Chem. 15, 899–916. Still, W.C., Tempczyk, A., Hawley, R.C., Hendrickson, T., 1990. Semianalytical treatment of solvation for molecular mechanics and dynamics. J. Am. Chem. Soc. 112, 6127–6129. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., Takeshige, K., 1994. Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. Proc. Natl. Acad. Sci. U.S.A. 91, 5345–5349. Sumimoto, H., Hata, K., Mizuki, K., Ito, T., Kage, Y., Sakaki, Y., Fukumaki, Y., Nakamura, M., Takeshige, K., 1996. Assembly and activation of the phagocyte NADPH oxidase. J. Biol. Chem. 271, 22152–22158. Thrasher, A.J., Keep, N.H., Wientjes, F., Segal, A.W., 1994. Chronic granulomatous disease. Biochim. Biophys. Acta 1227, 1–24, doi:10.1016/09254439(94)90100-7.
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Verlet, L., 1967. Computer “experiments” on classical fluids. I. Thermodynamical properties of Lennard–Jones molecules. Phys. Rev. 159, 98–103, doi:10.1103/PhysRev.159.98. Wallace, A.C., Laskowski, R.A., Thornton, J.M., 1995. LIGPLOT: A program to generate schematic diagrams of protein–ligand interactions. Protein Eng. 8, 127–134. Yuzawa, S., Suzuki, N.N., Fujioka, Y., Ogura, K., Sumimoto, H., Inagaki, F., 2003. Crystallization and preliminary crystallographic analysis of the auto-inhibited form of the tandem SH3 domain of p47phox . Acta Crystallogr. D 59, 1479–1480.
Yuzawa, S., Suzuki, N.N., Fujioka, Y., Ogura, K., Sumimoto, H., Inagaki, F., 2004. A molecular mechanism for auto-inhibition of the tandem SH3 domains of p47phox , the regulatory subunit of the phagocyte NADPH oxidase. Genes Cells 9, 443–456. Zhang, L., Gallicchio, E., Friesner, R., Levy, R.M., 2001. Solvent models for protein–ligand binding: comparison of implicit solvent Poisson and surface generalized Born models with explicit solvent simulations. J. Comput. Chem. 22, 591–607, doi:10.1002/jcc.1031.
Case study
Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47phox, regulating NADPH oxidase activity Yoko Watanabe a , Hideyuki Tsuboi a , Michihisa Koyama a , Momoji Kubo a,b , Carlos A. Del Carpio a , Ewa Broclawik c , Eiichiro Ichiishi c , Masahiro Kohno c , Akira Miyamoto a,c,∗ a
Department of Applied Chemistry, Graduate School of Engineering, Tohoku University, 6-6-11-1302 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan b PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan c New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan Received 16 June 2005; received in revised form 10 April 2006; accepted 24 April 2006
Abstract The phagocyte NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of superoxides. Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by the absence of certain components of the NADPH oxidase. Key to the activation of the NADPH oxidase is the cytoplasmic subunit p47phox , which includes the tandem SH3 domains (N-SH3 and C-SH3). In active phagocytes, p47phox forms a stable complex with the cytoplasmic region of membrane subunit p22phox that forms a left-handed polyproline type-II (PPII) helix conformation. In this report, we have analyzed the conformational changes of p47phox –p22phox complexes of wild-type and three mutants, which have been detected in CGD patients, using molecular dynamics simulations. We have found that in the wild-type, two basal planes of PPII prism in cytoplasmic region of p22phox interacted with N-SH3 and C-SH3. In contrast, in the modeled mutants, the residue at the ape of PPII helix, which interacts simultaneously with both of the tandem SH3 domains in the wild-type, moved toward C-SH3. Furthermore, interaction energies of the cytoplasmic region of p22phox with C-SH3 tend to decrease in these mutants. All these findings led us to conclude that interactions between N-SH3 of p47phox and PPII helix, which is formed by cytoplasmic region of p22phox , may play a significant role in the activation of the NADPH oxidase. © 2006 Elsevier Ltd. All rights reserved. Keywords: Molecular dynamics; NADPH oxidase; SH3 domain; PPII helix; Ligand recognition
The phagocyte NADPH oxidase is found primarily in neutrophils complex and plays a crucial role in the innate immune system that defends the host against bacterial, fungal, and viral pathogens. NADPH oxidase plays an important role in the defense system of human body by catalyzing the NADPH oxidation (Eq. (1)), which leads to the production of superoxide from oxygen and NADPH (Ago et al., 1999; Thrasher et al., 1994) NADPH + 2O2 → NADP+ + H+ + 2O2 −
∗
Corresponding author. Tel.: +81 22 795 7233; fax: +81 22 795 7235. E-mail addresses: [email protected] (A. Miyamoto), [email protected] (H. Tsuboi), [email protected] (M. Koyama), [email protected] (M. Kubo), [email protected] (C.A. Del Carpio), [email protected] (E. Broclawik), [email protected] (E. Ichiishi), [email protected] (M. Kohno). 1476-9271/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.compbiolchem.2006.04.004
(1)
Superoxides are converted, in turn, by means of enzymatic and non-enzymatic pathways to more potent oxidizing agents (Miller and Britigan, 1995). Neutrophils localize NADPH oxidases into the phagolysosomal membrane surrounding engulfed microbes which are killed by the oxidizing agents generated by NADPH oxidases. Inappropriate activation of this enzyme leads to the overproduction of superoxide, resulting in inflammatory responses and a number of disease states including chronic inflammation (Benard et al., 1999). Therefore, controlling superoxide production requires a detailed understanding of the activation mechanism of NADPH oxidase. In the resting state, the NADPH oxidase subunits are localized separately at the membrane and cytoplasm. The catalytic core of NADPH oxidase is the integral membrane protein cytochrome b558 that consists of gp91phox and p22phox subunits (Huang et al., 1995). However, cytochrome b558 is inactive in the absence of at least three cytoplasmic subunits p47phox , p67phox , and the small GTPase Rac that translocate to the membrane and
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Fig. 1. Domain organization of NADPH oxidase subunits (A) p47phox and (B) p22phox . PX, SH3, P, and T represent phox homology domain, Src homology domain 3, proline-rich region, and transmembrane domain, respectively.
bind cytochrome b558 during phagocytosis (DeLeo and Quinn, 1996). Key to this translocation process is p47phox , which consists of a PX domain, N-terminal SH3 domain (N-SH3), C-terminal SH3 domain (C-SH3), a polybasic region, and a proline-rich region (Fig. 1(A)). The tandem SH3 domains bind to the C-terminal cytoplasmic proline-rich region of p22phox (Fig. 1(B)), and this interaction is essential for NADPH oxidase activity (Sumimoto et al., 1996). In the inactive state, the binding surface of the tandem SH3 domains is masked through an intra-molecular interaction with polybasic region, resulting in the auto-inhibited form (Ago et al., 1999; Huang and Kleinberg, 1999; Sumimoto et al., 1994; Leto et al., 1994). In response to phagocyte activation, several serine residues located in the polybasic region of p47phox are phosphorylated, leading to changes in the conformation of p47phox and exposure of the binding surface of the tandem SH3 domains (Groemping et al., 2003; Ago et al., 2003). Chronic granulomatous disease (CGD) is caused by mutations in genes encoding the four PHOX subunits of NADPH oxidase (Casimir et al., 1992). Mutations in these genes cause a significant decrease in production of superoxide, and accordingly, CGD patients repeat life-threatening infections. Specifically, the gene for gp91phox , which is on the X chromosome, accounts for approximately 60% of all CGD cases. The rest of PHOX proteins, p22phox , p47phox , and p67phox , are autosomal and account for the remainder cases of CGD. Studies have shown that defects in p67phox and p22phox each account for only 5% of CGD patients (Roos et al., 1996). The remaining 30% of patients have defects in p47phox , indicating the importance of p47phox to NADPH oxidase activity. Recently, Groemping et al. (2003) determined crystal structure of the active state of p47phox , which forms a complex with the C-terminal cytoplasmic proline-rich region of p22phox , by multi-wavelength anomalous diffraction. Gly 192, 262 of p47phox and Pro 156 of p22phox are involved in this complex, and are important residues whose mutations cause CGD. Groemping et al. (2003) also measured the binding affinity of this complex in order to investigate the influences of mutation sites in p47phox –p22phox complex. Mutations in the SH3 domain of p47phox reduced the affinity for the p22phox . However, the influences of mutants on the structure of complex and on the inter-molecular interaction, which cause such reduction of binding affinity, remain to be elucidated. While computational chemistry approaches are effective to derive information at the atomic level, which cannot be easily obtained from experimental approach, to our knowledge, no computational chemistry study has been performed on this system so far.
In the present study we attempt to gain insights into the submolecular mechanisms triggering the production of superoxides using available computational chemical methods. To achieve this goal, we focus concretely on conformational changes that are induced by mutations in the involved proteins that are known to be the cause of CGD in humans. Three mutants, detected in CGD patients, are mainly studied in order to elucidate the factors that determine the superoxide-generating ability of NADPH oxidase. We simulated the dynamics of p47phox –p22phox complexes of the wild-type and three mutants, and investigated the plausible changes in the inter-molecular interactions between SH3 domains of p47phox and the cytoplasmic region of p22phox . The results derived by these simulations deem an important controlling role in the activity of NADPH oxidase to the interaction between N-SH3 of p47phox and the PPII helix region of p22phox . Consequently, NADPH oxidase may not be able to produce super oxides when p47phox does not form a complex with p22phox . Furthermore, lack of interaction between N-SH3 regions corresponding to p47phox and p22phox , which is induced by change of the positional relationship between the tandem SH3 domains and the PPII helix region of p22phox , seems to also affect the superoxide-generating ability of the system. 1. Methods In the present study all the calculations we used the OPLSAA force field (Jorgensen et al., 1996) for intra- and intermolecular interactions and the surface generalized Born (SGB) model (Zhang et al., 2001; Ghosh et al., 1998) for the solvent electrostatic effects as implemented in the IMPACT program (Schr¨odinger, Inc.). 1.1. OPLS-AA force field The OPLS-AA force field is known as a force field that well reproduces liquid state properties of molecules. This force field uses empirical experimental data from the liquid state and quantum mechanical calculations to determine parameters for intra-molecular bond, angle, and torsion motions to set the constituent parameters. The intra-molecular interaction is given as, Vintra = Kr (r − req )2 + Kθ (θ − θeq )2 + Vtorsion (2) bonds
angles
where Kr and Kθ represent the force constants, and r and θ are bond lengths and angles, respectively. Suffix eq means equilibrium value. Torsional interaction is assessed as given in, Vtorsion =
Vi
1
i
+
2
[1 + cos(ϕ)] +
V3i [1 + cos(3ϕ)] 2
V2i [1 − cos(2ϕ)] 2 (3)
where the torsional interaction is summed over all the dihedral angles ϕi , and V1 –V3 are the Fourier coefficients. The nonbonded interaction is given as a van der Waals term together
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with an electrostatic term, σij12 σij6 qi qj 4εij − 6 + Vinter = Rij R12 Rij ij i>j
D=
1.2. Surface generalized Born model Recently, extensive efforts have been directed to the development of implicit solvation models for simulations of biomolecular systems that are physically reasonable and simultaneously calculation cost-effective. In SGB models, which are based on approximations of the Poisson–Boltzmann equation, the solute is described in atomic detail, while the solvent is replaced by a dielectric continuum. The Born equation gives the electrostatic free energy of transferring a spherical charged ion from a medium of dielectric constant εi to a medium of dielectric constant εo (Born, 1920). For a real molecule with an arbitrary-shaped cavity formed by the molecular surface, the electrostatic free energy of solvation is given as follows n n qi qj 1 1 1 − GGB = − 2 εi εo rij2 + α2ij e−D i=1 j=1 1 2
1 − 2
Gpair
1 =− 2
Gsingle
1 =− 2
1 1 − εi εo 1 1 − εi εo
1 1 − εi εo
n n
qi qj 2 2 −D i=1 j=1 j=i rij + αij e
n i=1
qi2 = Gpair + Gsingle αi
n n
1 1 − εi εo
i=1 j=1 j=i
n i=1
qi2 αi
rij2
(11)
(2αij )2
(4)
where qi and qj represent permanent Coulomb charges, σ ij and εij are the Lennard–Jones parameters, Rij is the inter-atomic distance. The same expression is used for intra-molecular nonbonded interactions between all pairs of atoms separated by three or more bonds. The (1,4)-interactions are scaled by a factor of 1/2. The non-bonded parameters ε and σ for each atom-pair is constructed from the atomic values by the geometric mean combination rule, √ (5) εij = εi εj √ σij = σi σj (6)
=−
305
qi qj rij2 + α2ij e−D
(7)
(8)
(9)
Eq. (9) is called the generalized Born equation (Still et al., 1990), where q and α are charge and the radius of the atom, respectively, and rij is the distance between atoms i and j. Here, αij and D are expressed as Eqs. (10) and (11), respectively, √ (10) αij = αi αj
Gsingle is the self-energy, which has exactly the same form as the Born equation, and Gpair is the pair energy. In SGB model, the effective Born radius αi can be obtained from Eq. (9) and the following equation, ∆Gsin gle
1 =− 8π
1 1 − εi εo
i
× (R − ri ) n(R) d 2 R
s
qi2 |R − ri |4 (12)
Here, ri represents the coordinates of atom i, and n(R) represents the vector of integration over the surface of the molecule (R). Then the total electrostatic free energy of solvation can be obtained by the generalized Born equation (Eq. (7)) with the effective Born radius for every atom of the solute molecule. Further details of OPLS-AA and SGB models are available in (Jorgensen et al., 1996) and (Zhang et al., 2001; Ghosh et al., 1998), respectively. 1.3. Models and conditions X-ray crystal structure of p47phox complexed with p22phox was obtained from the Protein Data Bank (PDB code: 1OV3 (Groemping et al., 2003)). The initial structure for the wildtype was obtained by refining this X-ray crystal structure using molecular mechanics energy minimization including the reported crystal water molecules. The water molecules were then removed from the finally obtained structure. We determined the initial structure of three mutants, G192S, G262S, and P156Q, based on the initial structure of the wild-type using the rotamer library of Swiss-PDB viewer (Guex and Peitsch, 1997). In mutants G192S and G262S, Gly 192 and Gly 262 of p47phox are substituted to Ser, and in P156Q, Pro 156 of p22phox is substituted to Gln. Additional minimization around the mutation site was performed for these modeled mutants. In molecular dynamics simulations, the velocity form of the Verlet algorithm (Verlet, 1967) was used with a time step of 2.0 fs to integrate the equations of the motions. 500,000 steps MD calculations were performed at 310 K for each structure. The atoms located in the N- and C-terminal residues of p47phox , and in the N-terminal (cytosolic side) residue of p22phox were treated as “buffered”. Buffered atoms are allowed to move, subject to harmonic penalty-function restraints that tether them to their initial positions. 1.4. Visualization and analysis Molecular graphics images were created with UCSF Chimera code (Pettersen et al., 2004), and interactions between protein and ligand were analyzed by schematic representation using the LIGPLOT program (Wallace et al., 1995).
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2. Results and discussion 2.1. Validation of the initial structure and molecular dynamics simulations As mentioned above, we first performed structure optimization of the protein structure with crystal water molecules to determine the initial structure, because these molecules are important for protein stability. Fig. 2(A) shows the root mean square deviations (RMSD’s) between corresponding residues of the X-ray crystal structure and the obtained initial structure. RMSD’s values are small for the ␣-helix and -strand regions of the protein, which are indicated by arrows in Fig. 2(C), while RMSD’s values are large for the loop region. This is a sign that the initial structure was adequately generated from the X-ray crystal structure of p47phox –p22phox complex. In the successive molecular dynamics simulations, the crystal water molecules were removed and the SGB model was applied in order to evaluate the solvent effect. The adequacy of this approximation was confirmed by a meticulous estimation of the structural changes arisen on each model. Similarly as for the initial structure, RMSD values for corresponding residues in the initial and final structures are large in the loop region while the RMSD values are small in the ␣-helix and -strand regions. Moreover, none significant structural change in the wild-type (Fig. 2(B)) was observed, confirming thus the adequacy of the series of these simulations. 2.2. A novel mode of SH3–ligand interaction of p47phox In the active and inactive phagocyte, the SH3 domains of p47phox form a complex with the cytoplasmic region of p22phox and the polybasic region of p47phox , respectively. Both of these two ligands form a PPII helix conformation. Analysis of the initial structure, computed here by energy minimization, shows an agreement with this observation since the cytoplasmic region of p22phox interacts with both of N-SH3 and C-SH3 in the active phagocyte (Fig. 3). The binding of the tandem SH3 domains of p47phox and cytoplasmic region of p22phox is illustrated in
Fig. 2. Root mean square deviation (RMSD) of the initial and final structures of p47phox –p22phox complex against residue number. (A) RMSD of the initial structure from X-ray crystal structure. (B) RMSD of each final structure from the initial structure. Residues located in N-SH3 and C-SH3 of the final structure were superimposed on that of the initial structure, and then RMSD was calculated. (C) Secondary structure of p47phox . The arrows indicate ␣-helices and -strands.
Fig. 3. Inter-molecular interaction between the tandem SH3 domains of p47phox and cytoplasmic region of p22phox . The residues of cytoplasmic region of p22phox are shown in purple. The residues of N-SH3 and C-SH3 of p47phox were drawn in left side and right side of p22phox , respectively. Hydrogen bonds and hydrophobic interactions are indicated black dashed lines and green semi-circles, respectively.
Fig. 4(A). Residues 154–158 in p22phox form a PPII helix conformation that is characterized by a triangular prism-like structure. The basal plane formed by residues Asn 154, Pro 156 and Pro 157 interact with N-SH3, while the basal plane formed by Pro 155, Pro 156, and Arg 158 with C-SH3. Pro 156, located at the apex of the prism and formed by those basal planes, interacts with N-SH3 and C-SH3, simultaneously. Since this synergetic interaction, stabilizes the complex (as shown by our calculations of the initial structure), destabilization of the same can be assumed to be directly dependant to the formation or not of this particular spatial arrangement or conformation of the amino acids in the interacting subunits. And, by extension, this conformation can be thought to play an important role in the activation of p47phox . Our assumption is further supported by the fact that this interaction is radically different in other SH3 domain/target complexes. Moreover, molecular recognition between SH3 domain and PPII helix has been elucidated by X-ray crystallography and NMR, and is well characterized (Mayer, 2001; Kuriyan and Cowburn, 1997). These reported experimental observations stand for a PPII helix that takes on a structure analogical to a triangular prism, where residues at the single basal plane bind to a single SH3 domain, while those at the apex are exposed. In sharp contrast to these observations, Groemping et al. (2003) and Yuzawa et al. (2004) have found a different
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307
Fig. 4. Binding of the tandem SH3 domains of p47phox and its ligands that are forming PPII helix conformation. (A) Tandem SH3 domains of p47phox and cytoplasmic region of p22phox (residues 154–158) in the initial structure determined by structure optimization. (B) X-ray crystal structure of the tandem SH3 domains of p47phox and polybasic region of p47phox (residues 297–301) (Yuzawa et al., 2003).
mode of SH3–ligand interaction in the X-ray crystal structure of auto-inhibited p47phox , where the two basal planes of the PPII prism interact with N-SH3 and C-SH3 in auto-inhibited p47phox as shown in Fig. 4(B). This indicates that the polybasic region of p47phox does not bind tightly to each SH3 domain enabling in this way the ligand exchange with the cytoplasmic region of p22phox when p47phox is activated. On the contrary, auto-inhibited p47phox is stabilized by the synergic interaction between the tandem SH3 domains and polybasic region of p47phox (Groemping et al., 2003; Yuzawa et al., 2004). We found that the mode of inter-molecular interaction between the tandem SH3 domains of p47phox and the cytoplasmic region of p22phox observed in our simulations is the same as that of the intra-molecular interaction in p47phox whose X-ray structure has been reported in the literature (Groemping et al., 2003; Yuzawa et al., 2004). This leads to the conclusion that the same mode of interaction between the SH3 domain and its ligand is required for both of the active and inactive states of NADPH oxidase. 2.3. The role of the mutation sites in the activation of NADPH oxidase
between the two SH3 domains. On the contrary, the distance between Ser 262 and Ile 159 remained almost intact as well as the distance between Gly 262 and Ile 159 (Fig. 6(C)). In the initial structure of the wild-type, the sidechains of Trp 193 and Trp 263 form hydrogen bonds with the backbone carbonyl oxygen atoms of Asn 154 and Pro 156 of p22phox , connecting the cytoplasmic region of p22phox with N-SH3 and C-SH3 of p47phox , respectively (Fig. 5). While the mutation of Gly 262 to serine has almost no perceptible effect on the atomto-atom distance between Trp 263 and Pro 156 (Fig. 6(D)), the mutation of Gly 192 to serine increases these distances between Trp 193 and Asn 154 (Fig. 6(B)). In contrast, mutation of Pro 156 to glutamine affects the atom-to-atom distance between Trp 263 and Gln 156 (Fig. 6(D)). Although both, Gly 192 and 262 are residues in n-Src loop of p47phox , only structural changes in G262S are less severe than that in G192S. This result is in fair agreement with isothermal titration calorimetry measurements carried out by Groemping et al. (2003), which report that the affinity of p47phox with p22phox is reduced both in G192S and G262S, and the effect is more significant in G192S than in G262S. From our simulations, it can be inferred that the increased instability of G262S as compared with G192S is the result of the lack of intra-molecular interaction between the two
Since clinical data show the presence of particular mutants of p47phox as well as p22phox in patients with CGD, we carried out a further analysis of the plausible interaction patterns of the mutants and compared them with that in the wild type complex. Mutated structures of p47phox –p22phox complex were obtained using molecular dynamics simulations. In order to elucidate the structural changes caused by the mutations, we analyzed the hydrogen bonds appearing and disappearing between the mutated sites and the residues around them, as compared to those in the wild type complex. In the initial structure of the wild-type, which was determined by structure optimization, the amide nitrogen atom of Gly 192 and Gly 262 form hydrogen bond with the backbone carbonyl oxygen atom of Ser 189 and Ile 259, respectively (Fig. 5). These hydrogen bonds stabilize the n-Src loop in N-SH3 and C-SH3, which mediates the interaction between the two SH3 domains. When Gly 192 is mutated to serine, the distance between Ser 192 and Ser 189 increases (Fig. 6(A)), indicating that the mutation of Gly 192 to serine disrupts the n-Src loop of N-SH3 and consequently the interaction
Fig. 5. Hydrogen bonds formed by the mutation site or the residues around it. Hydrogen bonds are indicated with purple lines. The residues located in N-SH3, C-SH3 and p22phox are shown in light blue, light green, and pink.
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Fig. 6. The changes in atom–atom distance shown in Fig. 5. Atom–atom distances between (A) the amide nitrogen atom of Gly (Ser in G192S) 192 and the backbone carbonyl oxygen atom of Ser 189; (B) the sidechain of Trp 193 and the backbone carbonyl oxygen atom of Asn 154; (C) the amide nitrogen atom of Gly (Ser in G262S) 262 and the backbone carbonyl oxygen atom of Ile 259; (D) the sidechain of Trp 263 and the backbone carbonyl oxygen atom of Pro (Gln in P156Q) 156.
SH3 domains due to the disruption of the n-Src loop in N-SH3. Additionally, Pro 156 stabilizes the inter-molecular interaction between C-SH3 domain of p47phox and the cytoplasmic region of p22phox and is important to form p47phox –p22phox complex. 2.4. Inter-molecular interaction between p47phox and p22phox Mutation at Gly 192, Gly 262 of p47phox and Pro 156 of p22phox leads to a difference in the interaction energy of the cytoplasmic region of p22phox with the SH3 domains of p47phox . This difference was estimated computing the interaction energies for
the complexes using the following equation: Einteraction = Ecomplex − (Ereceptor + Eligand )
(13)
where Ecomplex is the total energy of the p47phox –p22phox complex, and Ereceptor and Eligand are respectively those of the receptors (tandem SH3 domains, N-SH3, and C-SH3) and ligands (SH3-binding region and PPII helix region). Complexes of N-SH3-p22phox and C-SH3-p22phox were minimized around p22phox before calculating the interaction energy. Fig. 7 shows the changes in interaction energy for the SH3-binding region of p22phox with the SH3 domains, and Table 1 shows the interaction energy for the PPII helix region of p22phox with the SH3
Table 1 Interaction energy of PPII helix region of p22phox with the SH3 domains of p47phox in the initial structures determined by structure optimization Model Wild-type G192S G262S P156Q Wild-type G192S G262S P156Q Wild-type G192S G262S P156Q
Receptor
Tandem SH3
N-SH3
C-SH3
Ligand
Interaction energy (kcal/mol)
PPII helix region of p22phox
−74.224 −56.911 −74.561 −69.111
PPII helix region of
p22phox
−23.944 −18.038 −23.831 −27.648
PPII helix region of
p22phox
−51.034 −50.768 −58.264 −42.015
Tandem SH3 domains, N-SH3, and C-SH3 correspond to residues 159–283, 159–213, and 229–283 of p47phox . SH3-binding region and PPII helix region correspond to residues 150–160 and 154–158 of p22phox .
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Fig. 7. Interaction energy of SH3-binding region of p22phox with the SH3 domains of p47phox during molecular dynamics simulation. (A) Wild-type, (B) G192S, (C) G262S, and (D) P156Q. Interaction energy of SH3-binding region with the tandem SH3, N-SH3 and C-SH3 are shown in gray, black, and white, respectively.
domains in the initial structure, respectively. While the interaction energy of the SH3-binding region with N-SH3 increases proportionally to the simulation time in the wild-type complex, interaction energies decrease in three of the mutants. On the contrary, interaction energies of SH3-binding region with the tandem SH3 domains and C-SH3 do not change significantly during these simulations. This can be considered an indication that the lack of interaction between N-SH3 of p47phox and the cytoplasmic region of p22phox induces inactivation of NADPH oxidase. In initial structures of all the investigated cases, which were determined by structure optimization, interaction energies of SH3-binding region with N-SH3 are almost the same as those with C-SH3 (Fig. 7). On the contrary, interaction energies of PPII helix region with C-SH3 have apparently less value than those with N-SH3 (Table 1), indicating stronger interaction. Therefore, we can conclude from our calculations that the cytoplasmic region of p22phox forms a stable complex with both of the tandem SH3 domains, and that the PPII helix region interacts mainly with C-SH3. Additionally this PPII helix conformation contributes significantly to the interaction between p47phox and p22phox , and can be deemed one of the important factors in the determination of the active/inactive state of NADPH oxidase.
mic region of p22phox . To analyze this hypothesis we performed further molecular dynamic simulations of this protein complex. Since molecular dynamics simulations produce large numbers of conformations for the wild-type and mutated p47phox –p22phox complexes, to study the structural changes in the cytoplasmic region of p22phox . We focused on the low energy conformations of these complexes. The total energy curves for the molecular dynamics simulations have local minimum values at several points. As shown in Fig. 8, the total energy of the wild-type has local minimum values at 520, 870, 910, and 950 ps, G192S at 740 and 870 ps, G262S at 400, 770, and 810 ps, P156Q at 250 and 300 ps. We considered that the structures at these time steps exist as stable conformations and thus analyzed
2.5. Structural changes of the cytoplasmic region of p22phox The observation of the decrement in interaction energy between N-SH3 of p47phox and the cytoplasmic region of p22phox in three of the analyzed mutants, may also be attributed to conformational change of the PPII helix region of the cytoplas-
Fig. 8. Total energy changes, including SGB energy, during the molecular dynamics simulations.
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them in detail. However, the relationship among all the generated structures cannot be neglected if a complete analysis of the problem is targeted, as is the case of the present work. Therefore, we performed a cluster analysis to filter minor conformations using the XCluster software (Shenkin and McDonald, 1994) which is a tool for observing and analyzing the molecular conformations based on molecular similarity criteria. Our analysis revealed that structures, which energy values similar to local minima structures are observed with high frequency during molecular dynamics simulations. This means that these structures exhibit stable conformations and are not just mere minor conformations. In order to infer the cause of decrease of interaction energy between N-SH3 of p47phox and the cytoplasmic region of p22phox in three mutants using computational instances alone, we analyzed the conformational change of the PPII helix region in the cytoplasmic region of p22phox (residues 154–158) using these low energy conformations. Before the conformational analyses we performed an energy minimization process around p22phox . Fig. 9 shows the Ramachandran plot for residues 154–158 in p22phox of low energy structures. Plots for the wildtype, G192S, and G262S are around the plot for typical PPII helix (Fig. 9(A)–(C)), meaning that these residues maintain the original PPII helix conformation during the molecular dynamics simulations. However, a difference in the positional relationship between the tandem SH3 domains and PPII helix region in the wild-type, G192S, and G262S mutants can be observed. Fig. 10 shows the structure of the tandem SH3 domains and PPII helix region after molecular dynamics simulation. In the wild-type,
Fig. 9. Ramachandran plots for PPII helix region of cytoplasmic region of p22phox (residues 154–158) during the molecular dynamics simulations. (A) Wild-type, (B) G192S, (C) G262S, and (D) P156Q. Phi and psi designate the rotation angle around the N–C␣ and C␣ –C bonds in polypeptide backbone, respectively. The blue solid lines and light blue regions indicate the range of allowed and core regions (Morris et al., 1992), respectively. The black solid line indicates the plot of typical PPII helix.
Fig. 10. The tandem SH3 domains of p47phox and cytoplasmic region of p22phox during molecular dynamics simulation. (A) Wild-type at 950 ps, (B) G192S at 870 ps, (C) G262S at 810 ps, and (D) P156Q at 300 ps. PPII helix region of p22phox (residues 154–158) and the mutation site are shown in pink and yellow, respectively.
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two basal planes of the PPII prism interact with both of the tandem SH3 domains (Fig. 10(A)) in the same manner as the initial structure determined by structure optimization. In contrast, the backbone of Pro 156 that interacted with both N-SH3 and CSH3 in the initial structure interacts only with C-SH3 in G192S and G262S (Fig. 10(B) and (C)). In P156Q, Ramachandran plot deviates from typical PPII helix, meaning that residues 154–158 cannot maintain PPII helix conformation during the molecular dynamics simulation (Fig. 9(D)). Moreover, P156Q shows no local minimum value after 300 ps. Therefore, we can hypothesize that P156Q cannot form a stable p47phox –p22phox complex, because the PPII helix conformation in cytoplasmic region of p22phox is disrupted. 3. Conclusions In the present study we used computational methodologies to investigate the most important structural factors of the system p47phox –p22phox that may be involved in the activation of NADPH and its superoxide-generating ability. Our study reveals that the SH3 domain may play an important role as the key mediator of interactions that regulate the formation of NADPH oxidase complex and enzyme activity. The calculations show that the cytoplasmic region of p22phox interacts synergetically with the tandem SH3 domains of p47phox , leading us to hypothesize that this results in the activation of NADPH oxidase. Our simulation results of p47phox –p22phox complexes for wild-type and mutants show the characteristics of this intermolecular interaction. Our simulation results also reveal that the PPII helix conformation of the cytoplasmic region of p22phox plays an indispensable role in the formation of p47phox –p22phox complex. In spite of the synergetic interaction in p47phox –p22phox complex, the PPII helix region interacted mainly with C-SH3. In all of the investigated mutants, that are not able to produce superoxide, interaction energies of SH3binding region with N-SH3 decrease. However, there is a difference in the inactivation mechanism of the investigated mutants. The cytoplasmic region of p22phox maintains PPII helix conformation, and synergic interaction between PPII helix region and tandem SH3 domains of p47phox is disrupted in G192S and G262S. On the contrary, the cytoplasmic region of p22phox cannot maintain PPII helix conformation in P156Q. The information obtained from our simulations and the subsequent analysis illustrates the inter-molecular interaction mode at the sub-molecular level between p47phox and p22phox that may be intimately related to the causes of inactivation of NADPH oxidase, and can be deemed of great importance for further advances in the medical treatment of CGD and chronic inflammation. References Ago, T., Nunoi, H., Ito, T., Sumimoto, H., 1999. Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47phox . J. Biol. Chem. 274, 33644–33653. Ago, T., Kuribayashi, F., Hiroaki, H., Takeya, R., Ito, T., Kohda, D., Sumimoto, H., 2003. Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides. Leading to phago-
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