p47phox molecular activation for assembly of the neutrophil NADPH oxidase complex.

The p47(phox) cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47(phox) activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22(phox) upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22(phox), reflecting an intermediate conformation between the autoinhibited and activated forms.