The active N-terminal region of p67phox: structure at 1.8 A resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease

Abstract Upon activation, the NADPH oxidase from neutrophils produces superoxide anions in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67phox, p47phox, and the small G protein Rac with a membrane-associated flavocytochrome b558. Here we report the crystal structure of the active N-terminal fragment of p67phox at 1.8 A resolution, as well as functional studies of p67phox mutants. This N-terminal region (residues 1–213) consists mainly of four TPR (tetratricopeptide repeat) motifs in which the C terminus folds back into a hydrophobic groove formed by the TPR domain. The structure is very similar to that of the inactive truncated form of p67phox bound to the small G protein Rac previously reported, but differs by the presence of a short C-terminal helix (residues 187–193) that might be part of the activation domain. All p67phox mutants responsible for Chronic Granulomatous Disease (CGD), a severe defect of NADPH oxidase function, are localized in the N-terminal region. We investigated two CGD mutations, G78E and A128V. Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro at 25 °C. However, this point mutation represents a temperature-sensitive defect in p67phox that explains its phenotype at physiological temperature.