Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production [Enzymology]

June 26th, 2014 by Meijles, D. N., Fan, L. M., Howlin, B. J., Li, J.-M.

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defence against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. S379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem SH3 domains, allowing the AIR to undergo phosphorylation to expose the tandem SH3 groove for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox-/- coronary microvascular cells. Compared to wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial superoxide production in response to acute PMA (a potent PKC activator) stimulation. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. S379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.