NMR Solution Structure of the Tandem Src Homology 3 Domains of p47 super(phox) Complexed with a p22 super(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 a multisubunit enzyme composed of membrane-bound flavocytochromeb sub(558) as well as cytosolic components, including p47 super(phox), which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b sub(558) due to binding of the tandem Src homology 3 (SH3) domains of p47 super(phox) to a proline-rich region in p22 super(phox), a subunit of flavocytochrome b sub(558). Using NMR titration, we first identified the proline-rich region of p22 super(phox) that is essential for binding to the tandem SH3 domains of p47 super(phox). We subsequently determined the solution structure of the p47 super(phox) tandem SH3 domains complexed with the proline-rich peptide of p22 super(phox) using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22 super(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 alpha -helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal alpha -helical region of the p22 super(phox) peptide increases the binding affinity for the tandem SH3 domains of p47 super(phox) more than 10-fold.