Atypical membrane-embedded phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2)-binding site on p47phox phox homology (PX) domain revealed by NMR

Abstract

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47phox PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P2 binding mode of the p47 phox PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P2-binding site, which includes the residues of helices α1 and α1′ and the following loop up to the distorted left-handed PPII helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47phox PX domain suggest that different types of phosphoinositides sequentially bind to the p47phox PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.