Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47phox Phox Homology (PX) Domain Revealed by NMR

Pavlos Stampoulis,Takumi Ueda,Masahiko Matsumoto,Hiroaki Terasawa,Kei Miyano,Hideki Sumimoto,Ichio Shimada

doi:10.1074/jbc.m111.332874

Abstract

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) 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)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) 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 p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.

Highlights

The p47phox Phox homology (PX) domain binds PI[3,4]P2 during assembly and activation of NADPH oxidase
In the Transferred cross-saturation (TCS) experiment, the complex is irradiated at a frequency centered at the aliphatic proton resonances, which exclusively affects the liposomes, because almost no aliphatic protons exist in the deuterated PX domain
The saturation is not limited within the liposomes but is transferred to the residues at the interface of the PX domain bound to the liposomes, through intermolecular cross-relaxation

Summary

Background

The p47phox PX domain binds PI[3,4]P2 during assembly and activation of NADPH oxidase. In the crystal structure of the CISK PX domain, one of the two bound sulfate groups was found in a completely unexpected area, which probably represents a nonspecific binding site [15]. It is unclear whether these sulfate-binding sites of p47phox PX are the actual ligand-binding sites, and the mechanism of the broad ligand specificity of p47phox PX is still unclear. Our data indicate that the p47phox PX domain binds PI[3,4]P2 in a unique area, as compared with the phosphoinositide-binding sites of all of the other PX domains characterized far (30 –35)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION