Binding of p67 phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369 Cys-Gly-Cys 371 triad in Nox2 and in p67 phox

Tanya Fradin, Edna Bechor, Yevgeny Berdichevsky, Iris Dahan, Edgar Pick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

A central event in the activation of the phagocyte NADPH oxidase involves binding of p67 phox to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67 phox to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357–383, as a potential binding site. A key role was attributed to a 369 Cys-Gly-Cys 371 triad, shared by peptides 357–371 (peptide 24) and 369–383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67 phox ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67 phox ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67 phox with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67 phox to serines abolishes binding and diminishes the ability of p67 phox to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67 phox with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the 369 Cys-Gly-Cys 371 triad and cysteine(s) in p67 phox .

Original languageEnglish
Pages (from-to)1023-1039
Number of pages17
JournalJournal of Leukocyte Biology
Volume104
Issue number5
DOIs
StatePublished - Nov 2018

Keywords

  • NADPH oxidase
  • cysteine oxidation
  • flavins
  • flavocytochrome b
  • peptide–protein interaction
  • synthetic peptides

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