p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond—Canaan sighted?

Abstract

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA–Nox2 fusion proteins to p67phox. We found, serendipitously, that deletion of residues 259–279 in p67phox (p67phoxΔ(259–279)), endowed it with the ability to bind selectively to Nox2 peptide 369–383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375–383. Binding of p67phoxΔ(259–279) to peptide 28 was mimicked by that of a (p67phox‐RacGTP) chimera. Both p67phoxΔ(259–279) and the (p67pho–RacGTP) chimera bound a NusA–Nox2 fusion protein, comprising residues 375–383. Specific single residue deletion mutants, within the p67phox sequence 259–279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259–279 sequence in p67phox, were found to autobind p67phox, suggesting that an intramolecular bond exists in p67phox, one pole of which was located within residues 259–279. We conclude that “resting” p67phox exists in a “closed” conformation, generated by an intramolecular bond. Deletion of specific residues within the 259–279 sequence, in vitro, or interaction with RacGTP, in vivo, causes “opening” of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

p67phox-derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism

“…In preliminary work, described in a recent publication, overlapping synthetic peptides, representing residues 1-300 in p67 phox , were tested for the ability to interfere with oxidase activity in a cell-free system. 32 This revealed the oxidase inhibitory property of a cluster of three peptides representing residues 259-279, located in SH3-N. In the present study, we followed up on these results by an in-depth analysis of the characteristics of the relevant peptides and by focusing on the mechanism underlying their inhibitory action.…”

“…An alternative explanation was offered by our recently reported finding that residues in the sequence 259-279 were engaged in an auto-inhibitory intramolecular bond with a yet unidentified region in p67 phox . 32 This led to the hypothesis that p67 phox peptides inhibit oxidase activity by a mechanism mimicking auto-inhibition. In the present study, we show that oxidase inhibition is mediated by peptides that self-assemble spontaneously.…”

“…The rationale for the present method of assessing the ability of p67 phox peptides to inhibit oxidase activity is rooted in the recent finding of an intramolecular bond in p67 phox responsible for an auto-inhibited state, 32 supported by the finding that peptides mapping at p67 phox residues 259-279 were capable of binding p67 phox in a protein-peptide binding assay. 32,42 In initial experiments, the same peptides were found to inhibit oxidase activity, using an assay protocol based on the assumption that inhibition was the result of competition between p67 phox peptides and p67 phox protein for binding to Nox2. We thoroughly modi- Figure S1.…”

“…On the basis of our recent finding that a region in SH3-N of p67 phox was engaged in an intramolecular bond and that residues NIVFVL played a key role in the process, 32 we hypothesized that self-assembled F I G U R E 8 Self-assembled p67 phox peptide 265-270 and its scrambled form inhibit NADPH oxidase activity by specific absorption of p67 phox . (A) Peptide 265-270 and its scrambled form (25 M) were incubated with a mixture of p47 phox , p67 phox , and Rac1 Q61L (100 nM, each) or with macrophage membrane liposomes (5 nM cytochrome b 558 heme) in cell-free assay buffer for 15 min at room temperature.…”

Reactive oxygen species are at the heart of innate immunity

“…Most recently, Professor Pick has focused on the interaction between the cytosolic component p67 phox , and Nox2 7,8 . The evidence gathered showed that, at the molecular level, p67 phox changes from a "resting" to an "activated" state when ROS production is required.…”