The molecular basis of Rac-GTP action-promoting binding of p67phox to Nox2 by disengaging the ß hairpin from downstream residues.

p67phox fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67phox to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67phox peptides (106-120) and (181-195), corresponding to the ß hairpin and to a downstream region engaged in intramolecular bonds with the ß hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67phox to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67phox , expressed in increased apparent molecular weights; (iv) p67phox was bound to p67phox peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67phox failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67phox -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67phox deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers. The molecular basis of Rac-GTP action entails unmasking of a previously hidden Nox2-binding site in p67phox , following disengagement of the ß hairpin from more C-terminal residues. The domain in Nox2 binding the "modified" p67phox comprises residues within the 369-383 sequence in the cytosolic dehydrogenase region.

p67phox -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism.

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67phox . In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67phox residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67phox , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition. In this study, we found that: (i) progressive N- and C-terminal truncation of inhibitory p67phox peptides, corresponding to residues 259-273 and 265-279, revealed that inhibitory ability correlated with the presence of residues 265 NIVFVL270 , exposed at either the N- or C-termini of the peptides; (ii) inhibition of oxidase activity was associated exclusively with self-assembled peptides, which pelleted upon centrifugation at 12,000 ×g; (iii) self-assembled p67phox peptides inhibited oxidase activity by specific binding of p67phox and the ensuing depletion of this component, essential for interaction with Nox2; and (iv) peptides subjected to scrambling or reversing the order of residues in NIVFVL retained the propensity for self-assembly, oxidase inhibitory ability, and specific binding of p67phox , indicating that the dominant parameter was the hydrophobic character of five of the six residues. This appears to be the first description of inhibition of oxidase activity by self-assembled peptides derived from an oxidase component, acting by an auto-inhibitory mechanism.

p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond-Canaan sighted?

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA-Nox2 fusion proteins to p67phox . We found, serendipitously, that deletion of residues 259-279 in p67phox (p67phox Δ(259-279)), endowed it with the ability to bind selectively to Nox2 peptide 369-383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375-383. Binding of p67phox Δ(259-279) to peptide 28 was mimicked by that of a (p67phox -RacGTP) chimera. Both p67phox Δ(259-279) and the (p67pho -RacGTP) chimera bound a NusA-Nox2 fusion protein, comprising residues 375-383. Specific single residue deletion mutants, within the p67phox sequence 259-279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259-279 sequence in p67phox , were found to autobind p67phox , suggesting that an intramolecular bond exists in p67phox , one pole of which was located within residues 259-279. We conclude that "resting" p67phox exists in a "closed" conformation, generated by an intramolecular bond. Deletion of specific residues within the 259-279 sequence, in vitro, or interaction with RacGTP, in vivo, causes "opening" of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 (phox.).

The superoxide (O(·-) 2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b 558 (a heterodimer of Nox2 and p22 (phox) ), and four cytosolic components, p47 (phox) , p67 (phox) , p40 (phox) , and Rac. The catalytic component, responsible for O(·-) 2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67 (phox) . Using a peptide-protein binding assay, we found that Nox2 peptides containing a (369)CysGlyCys(371) triad (CGC) bound p67 (phox) with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67 (phox) only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67 (phox) via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67 (phox) , in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67 (phox) to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.