Architecture of the p40-p47-p67phox complex in the resting state of the NADPH oxidase. A central role for p67phox.
The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67(phox) complex of the resting oxidase. Here we show that this complex contains one copy of each protein, which assembles to form a heterotrimeric complex. The apparent high molecular weight of this complex, as observed by gel filtration studies, is due to an extended, non-globular shape rather than to the presence of multiple copies of any of the proteins. Isothermal titration calorimetry measurements of the interactions between the individual components of this complex demonstrate that p67(phox) is the primary binding partner of p47(phox) in the resting state. These findings, in combination with earlier reports, allow us to propose a model for the architecture of the resting complex in which p67(phox) acts as the bridging molecule that connects p40(phox) and p47(phox).
Lapouge
Karine
K
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Smith
Susan J M
SJ
Groemping
Yvonne
Y
Rittinger
Katrin
K
D020816:Amino Acid Motifs; D002151:Calorimetry; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D008027:Light; D008957:Models, Genetic; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism; D010587:Phagocytosis; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013816:Thermodynamics; D014461:Ultracentrifugation
D010750:Phosphoproteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1