The active N-terminal region of p67(phox) - Structure at 1.8 angstrom resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease

Upon activation, the NADPH oxidase from neutrophils produces superoxide ani ons in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67(phox), p47(phox), and the smal l G protein Rac with a membrane-associated flavocytochrome b(558). Here we report the crystal structure of the active N-terminal fragment of p67(phox) at 1.8 Angstrom resolution, as well as functional studies of p67(phox) mut ants. 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 p67(phox) bound to the small C: p rotein Rac previously reported, but differs by the presence of a short C-te rminal helix (residues 187-193) that might be part of the activation domain . All p67(phox) mutants responsible for Chronic Granulomatous Disease (CGD) , a severe defect of NADPH oxidase function, are localized in the N-termina l region. We investigated two CGD mutations, G78E and A128V, Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro a t 25 degreesC, However, this point mutation represents a temperature-sensit ive defect in p67(phox) that explains its phenotype at physiological temper ature.