A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox

Iris Dahan, Susan M.E. Smith, Edgar Pick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

p67phox is the paramount cytosolic regulator of the superoxide-generating Nox of phagocytes, by controlling the conformation of the catalytic component, Nox2. The initiating event of this process is a protein–protein interaction between p67phox and the part of Nox2 protruding into the cytosol, known as the dehydrogenase region. The aim of this study was to identify and characterize region(s) in Nox2 acting as binding site(s) for p67phox. For this purpose, we measured the binding of recombinant p67phox to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288–570). We found that: 1) p67phox binds to a site corresponding to residues 357–383, represented by a cluster of 5 peptides (Nos. 24–28); 2) maximal binding was to peptides 24 (357–371) and 28 (369–383); 3) these shared a369Cys-Gly-Cys371 triad, found to be responsible for binding; 4) the Cys-Gly-Cys triad was present in Nox2 of mammals, birds, and amphibians but was absent in other Nox; 5) substituting a Nox4 or Nox1 sequence for the Nox2 sequence in peptide 24 abolished binding; 6) replacing 369Cys by Arg in peptide 24 (mimicking a mutation in chronic granulomatous disease) abolished binding; 7) the same replacement in peptide 28 did not affect binding, indicating the existence of an additional binding site. Our results reveal an essential role for the Cys-Gly-Cys triad in Nox2 in binding p67phox, seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67phox.

Original languageEnglish
Pages (from-to)859-874
Number of pages16
JournalJournal of Leukocyte Biology
Volume98
Issue number5
DOIs
StatePublished - Nov 2015

Keywords

  • Chronic granulomatous disease
  • Flavocytochrome b
  • NADPH oxidase
  • Peptide arrays
  • Superoxide

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