Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47(phox) in the association with p40(phox)

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox) and p40(phox) as well as by the Rac1-Rho-GDI heterodi mer. The regulation is a consequence of protein-protein interactions involv ing a variety of protein domains that are well characterized in signal tran sduction. We have studied the behavior of the NADPH oxidase cytosolic facto rs in solution using small angle neutron scattering and gel filtration. p47 (phox), two truncated forms of p47(phox), namely, p47(phox) without its C-t erminal end (residues 1-358) and p47(phox) without its N-terminal end (resi dues 147-390), and p40(phox) were found to be monomeric in solution. The di meric form of p67(phox) previously observed by gel filtration experiments w as confirmed. Our small angle neutron scattering experiments show that p40( phox) binds to the full-length p47(phox) in solution in the absence of phos phorylation, We demonstrated that the C-terminal end of p47(phox) is essent ial in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich reg ion of p47(phox). The radii of gyration observed for p47(phox) and the trun cated forms of p47(phox) (without the C-terminal end or without the N-termi nal end) show that all these molecules are elongated and that the N-termina l end of p47(phox) is globular. These results suggest that the role of amph iphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(pho x)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).