NMR solution structure of the tandem Src homology 3 domains of p47 ^phox complexed with a p22^phox-derived proline-rich peptide

The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is amultisubunit enzyme composed of membrane-bound flavocytochrome b₅₅₈ as well as cytosolic components, including p47^phox, which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b₅₅₈ due to binding of the tandem Src homology 3 (SH3) domains of p47^phox to a proline-rich region in p22^phox, a subunit of flavocytochrome b ₅₅₈. Using NMR titration, we first identified the proline-rich region of p22^phox that is essential for binding to the tandem SH3 domains of p47^phox. We subsequently determined the solution structure of the p47^phox tandem SH3 domains complexed with the proline-rich peptide of p22^phox using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is amonomer. The central region of the p22^phox peptide forms a polyproline type II helix that is sandwiched by the N- and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short α-helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal α-helical region of the p22^phox peptide increases the binding affinity for the tandem SH3 domains of p47^phox more than 10-fold.