Specificity of p47^phox SH3 Domain Interactions in NADPH Oxidase Assembly and Activation

Abstract

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47^phox, p67^phox, and p22^phox). p47^phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22^phox and cytosolic p67^phox. To understand the specific roles of each SH3 domain of p47^phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr¹⁶⁷ or Tyr²³⁷→Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47^phox-p22^phox interaction and plays a predominant role in oxidase activity and p47^phox membrane assembly, while the second p47^phox SH3 domain interacts with the NH₂-terminal domain of p67^phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47^phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22^phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.