The molecular basis of Rac-GTP action-promoting binding of p67 to Nox2 by disengaging the β hairpin from downstream residues.

p67 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 p67 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 p67 peptides (106-120) and (181-195), corresponding to the β hairpin and to a downstream region engaged in intramolecular bonds with the β hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67 to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67 , expressed in increased apparent molecular weights; (iv) p67 was bound to p67 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) p67 failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67 -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67 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 p67 , following disengagement of the β hairpin from more C-terminal residues. The domain in Nox2 binding the "modified" p67 comprises residues within the 369-383 sequence in the cytosolic dehydrogenase region.