p67^phox 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 p67^phox and there is evidence for a change in the configuration of p67^phox being required for binding to Nox2. To study this, we measured binding of p67^phox to a library of Nox2 peptides and binding of NusA–Nox2 fusion proteins to p67^phox. We found, serendipitously, that deletion of residues 259–279 in p67^phox (p67^phoxΔ(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 p67^phoxΔ(259–279) to peptide 28 was mimicked by that of a (p67^phox-RacGTP) chimera. Both p67^phoxΔ(259–279) and the (p67^pho–RacGTP) chimera bound a NusA–Nox2 fusion protein, comprising residues 375–383. Specific single residue deletion mutants, within the p67^phox sequence 259–279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259–279 sequence in p67^phox, were found to autobind p67^phox, suggesting that an intramolecular bond exists in p67^phox, one pole of which was located within residues 259–279. We conclude that “resting” p67^phox 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 p67^phox to a specific, previously unknown, site in Nox2.