The Domain Organization of p67^phox, a Protein Required for Activation of the Superoxide-Producing NADPH Oxidase in Phagocytes

Abstract

The phagocyte NADPH oxidase, crucial for innate immunity, is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. In activation of the oxidase, the multidomain protein p67^phoxplays a central role: it translocates to the membrane as a ternary complex with p47^phoxand p40^phox, and interacts with the small GTPase Rac to assemble with the membrane-integrated catalytic protein gp91^phox, leading to superoxide production. Here we show, using small-angle X-ray scattering (SAXS) analysis, that p67^phoxadopts an elongated conformation when it exists not only as a monomer but also as the heterotrimer. Although p67^phoxharbors an N-terminal TPR domain for binding to Rac and a p40^phox-interacting PB1 domain, followed by an SH3 domain that associates with p47^phox, the present model suggests that no or few apparent associations occur between the domains. The positions of the protein-interaction domains in p67^phoxcontribute to activation of the phagocyte NADPH oxidase: the first SH3 domain that is located between the TPR and PB1 domains positively regulates oxidase activation only when it is present at the correct position; the PB1 domain placed at this SH3 domain position inhibits the oxidase by interacting with p40^phox.

A Conserved Region between the TPR and Activation Domains of p67 Participates in Activation of the Phagocyte NADPH Oxidase

“…Mutations leading to the indicated amino acid substitutions in p67 phox and Noxa1 were introduced by PCR-mediated site-directed mutagenesis. The cDNAs were ligated to the following expression vectors: pGEX-6P (GE Healthcare Biosciences) for expression of proteins fused to GST in Escherichia coli; pcDNA3 (Invitrogen) for expression of Nox proteins in CHO cells; pEF-BOS for expression of HA-, FLAG-, or Myc-tagged proteins in CHO cells (22,23,27). All of the constructs were sequenced for confirmation of their identities.…”

“…After being washed with Hepes-buffered saline (120 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM MgCl 2 , 1 mM CaCl 2 , and 17 mM Hepes, pH 7.4), the cells were resuspended at a concentration of 1 ϫ 10 6 cells/ml in the buffer and tested for estimation of superoxide production. The superoxide-producing activity was determined by superoxide dismutase-inhibitable chemiluminescence with an enhancer-containing luminol-based detection system (Diogenes; National Diagnostics), as described previously (22,23,27). After the addition of the enhanced luminolbased substrate, the cells were preincubated for 5 min at 37°C and subsequently stimulated at the same temperature with or without 200 ng/ml of phorbol 12-myristate 13-acetate (Research Biochemicals International).…”

“…Although the target for the N-terminal SH3 domain remains unidentified, it likely enhances oxidase activation, which may be mediated by facilitating interaction of p67 phox with gp91 phox / Nox2 (22). Such domain arrangement is crucial for p67 phox to efficiently activate the phagocyte oxidase (23).…”

The Nox Family of NADPH Oxidases that Deliberately Produce Reactive Oxygen Species

“…3a). In contrast to p47 phox , p67 phox appears to adopt an elongated conformation with no or little apparent associations between the domains [14]. Although p40 phox is dispensable for gp91 phox /Nox2 activation, it constitutively associates with p67 phox via the PB1-PB1 interaction [15] to facilitate translocation to membranes, especially phagosomal membranes [1][2][3][4][5][6].…”

NADPH Oxidases in Innate Immunity

“…Making use of a biophysical approach, Yuzawa et al [10] utilized small-angle X-ray scattering analysis to evaluate the conformation of the Nox2 cytosolic regulatory activator protein, p67…”