Conformational changes in p47phox upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis

AbstractThe neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47phox promotes its translocation with p67phox and p40phox, followed by membrane interaction and assembly with flavocytochrome b558 into a functional complex. To characterise p47phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain