title: Conformational changes in p47(phox) upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis.
abstract: The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47(phox) promotes its translocation with p67(phox) and p40(phox), followed by membrane interaction and assembly with flavocytochrome b(558) into a functional complex. To characterise p47(phox) 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 p47(phox) 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.
authors.affiliation: Laboratoire des Protéines Membranaires, CEA, DSV, Institut de Biologie Structurale (IBS), 41 rue Jules Horowitz, Grenoble F-38027, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Marcoux
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: Mathieu
authors.initials: M
authors.lastname: Castellan
authors.forename: Corinne
authors.initials: C
authors.lastname: Vivès
authors.forename: Eric
authors.initials: E
authors.lastname: Forest
authors.forename: Franck
authors.initials: F
authors.lastname: Fieschi
mesh_terms: D041961:Deuterium Exchange Measurement; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1