Structural profiles of the full phagocyte NADPH oxidase unveiled by combining computational biology and experimental knowledge.

Abstract

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47^phox, p40^phox, p67^phox, and Rac) with the membrane-bound flavocytochrome b₅₅₈ (cytb₅₅₈). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb₅₅₈, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47^phox with cytb₅₅₈, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C-terminus of p47^phox interacts with the cytb₅₅₈. Combining the AF2 cytb₅₅₈ -p47^phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47^phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47^phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb₅₅₈-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multi-subunit assembly.