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

Edna Bechor, Anat Zahavi, Maya Amichay, Tanya Fradin, Aya Federman, Yevgeny Berdichevsky, Edgar Pick

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)509-528
Number of pages20
JournalJournal of Leukocyte Biology
Volume107
Issue number3
DOIs
StatePublished - 1 Mar 2020

Keywords

  • (p67-Rac) chimera
  • NADPH oxidase
  • NusA–Nox2 fusion protein
  • intramolecular bond
  • peptide–protein interaction
  • synthetic peptides

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