NADPH oxidase (NOX) is a multienzymatic complex found in many cellular types, responsible for reactive oxygen species (ROS) production. The neutrophilic NOX is composed of two transmembrane proteins (gp91phox and p22phox) that make up the catalytic core and three cytosolic factors (p47phox, p67phox and p40phox). Upon activation, p47phox, undergoes conformational changes that this study attempts to define in order to better understand the regulation of this complex involved in many diseases. In neutrophils, ROS are responsible for phagocyted pathogen destruction. It therefore appears essential to better understand the molecular bases of the NOX activation mechanism to consider its future regulation. This study has identified a number of conformational changes on p47phox by limited proteolysis and Deuterium eXchange coupled to Mass Spectrometry (DXMS). The AIR release, providing better accessibility to the p22phox binding site was confirmed and characterized from both structural and functional points of view, on the entire protein. Furthermore, a novel surface controlling p47phox auto-inhibited state has been discovered. Site-directed mutagenesis within this surface confirmed this hypothesis by identifying two key residues (R162 and D166) responsible for this auto-inhibition and therefore possible future candidates for therapeutic targets. The relative binding properties of these mutants with GST-p22phoxCter and liposomes were investigated using BiacoreTM and pull-down assays, respectively. The identification of these residues provided a better understanding of the p47phox activation mechanism, and in particular of how AIR unmasking leads to PX domain release. Finally, a methodological study showed that plasmepsin 2 from Plasmodium falciparum was a new tool that may improve DXMS.
