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Review
. 2005 Mar 15;386(Pt 3):401-16.
doi: 10.1042/BJ20041835.

Activation and assembly of the NADPH oxidase: a structural perspective

Yvonne Groemping  1 , Katrin Rittinger
Affiliations
Review

Activation and assembly of the NADPH oxidase: a structural perspective

Yvonne Groemping et al. Biochem J. .

Abstract

The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex. Multiple inputs are required to disrupt these inhibitory interactions and allow translocation to the membrane and association with the integral membrane components. Protein interaction modules are key regulators of NADPH oxidase assembly, and the protein-protein interactions mediated via these domains have been the target of numerous studies. Many models have been put forward to describe the intricate network of reversible protein interactions that regulate the activity of this enzyme, but an all-encompassing model has so far been elusive. An important step towards an understanding of the molecular basis of NADPH oxidase assembly and activity has been the recent solution of the three-dimensional structures of some of the oxidase components. We will discuss these structures in the present review and attempt to reconcile some of the conflicting models on the basis of the structural information available.

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Figures

Figure 1
Figure 1. Model of the cytochrome b558
The predicted transmembrane helices of gp91phox and p22phox are indicated. Glycosylation sites are indicated by cyan dots and regions that are believed to interact with p47phox in the active state in red. The FAD- and NADPH-binding sites in gp91phox are shown in cyan and pink respectively. The position of the consensus PxxP motif in the cytoplasmic region of p22phox that interacts with p47phox is indicated in grey.
Figure 2
Figure 2. Domain structure of the cytosolic subunits p40phox, p47phox and p67phox
The domain structure of the cytoplasmic components is shown as predicted by SMART (http://smart.embl-heidelberg.de/). The positions of consensus PxxP motifs in p47phox (amino acids 363–368) and p67phox (amino acids 226–236) are indicated by thick black bars. The locations of serine and threonine residues that become phosphorylated during activation are indicated by thin black bars.
Figure 3
Figure 3. Complex between the PB1 domains of p40phox and p67phox
Ribbons representation of the complex between the PB1 domains of p40phox in blue and p67phox in magenta (PDB code 1OEY) [119]. Residues from the basic clusters (BC1 and BC2) and acidic clusters (AC1 and AC2), which mediate complex formation are indicated in a ball and stick representation.
Figure 4
Figure 4. C-terminal SH3 domain of p67phox in complex with the C-terminal region of p47phox
p67-SH3B is shown in blue in a ribbon representation, while the C-terminal region of p47phox encompassing amino acids 359–390 is shown in a surface representation with underlying ribbons (PDB code 1KU4) [136]. The side chains of Ser359, Ser370 and Ser379, which become phosphorylated during activation, and of Arg368, Ile374 and Thr382, which are important for complex formation, are shown in a ball and stick representation. Furthermore, the positions of Pro363 and Pro366, which are part of the consensus PxxP motif are indicated.
Figure 5
Figure 5. Regulation of p47phox activity
Left-hand side: in the auto-inhibited conformation of p47phox, the tandem SH3 domains (blue) are masked by an intramolecular interaction with the polybasic region (yellow) where a core region interacts with conserved Trp193 and Trp263 residues (red) (PDB code 1NG2) [144]. The positions of serine residues 303, 304 and 328, which are important for activation, are indicated in red. The auto-inhibited core of p47phox crystallized as a domain-swapped dimer, but the monomer is shown in this Figure. Right-hand side: a proline-rich peptide from the cytoplasmic region of p22phox encompassing residues 151–160 (yellow) binds to the same SuperSH3 arrangement as the core of the polybasic region (PDB code 1OV3) [144]. In addition to the consensus PxxP core that is involved in hydrophobic contacts with both conserved ligand-binding surfaces of the SH3 domains (blue), there are extensive additional contacts between SH3A and the p22 peptide.
Figure 6
Figure 6. Model of the resting state of the NADPH oxidase
A model for the protein–protein interactions formed during the resting state of the NADPH oxidase, based on available X-ray and NMR structures (PDB codes 1O7K, 1NG2, 1KU4, 1OEY, 1E96 and 1H6H). The structures of the SH3 domains of p67phox (SH3A) and of p40phox have not yet been solved, nor have their binding partners been identified. In their place, a surface representation of the structure of an archetypal SH3 domain is shown in transparent grey (PDB code 2SEM). The positions of the activation domain (AD) and PxxP motif in p67phox are indicated in red and black respectively. At present, the molecular details of the cross-talk between the PX domain and tandem SH3 domains of p47phox are not understood (see text). This is represented by the grey and orange circles on top of the PX and SuperSH3 domains of p47phox.
Figure 7
Figure 7. Complex formation between Rac and p67phox
Complex formation between the active form of Rac in blue and the TPR domain of p67phox in orange occurs at the membrane (PDB code 1E96) [73]. The β-hairpin insertion, which contains Arg102 that is crucial for complex formation, is highlighted in yellow. Switch I, switch II and the insertion helix of Rac are highlighted in light green. The positions of Ala27 and Gly30, which determine specificity, and of Asp150, which appears to be important for subcellular localization, are indicated by red balls.
Figure 8
Figure 8. The PX domain as a phosphoinositide-binding module
The structure of the PX domain of p40phox bound to di-C4-PtdIns(3)P in a ribbon representation (PDB code 1H6H) [189]. Helix α1, which is important for solubility, but not part of the PX domain, is coloured blue. The phospholipid is shown in a ball-and-stick representation. The PPII helix is highlighted in pink. The positions of residues Tyr59, Arg85 and Arg105, which make important contacts with di-C4-PtdIns(3)P are indicated.
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