title: A Cys-Gly-Cys triad in the dehydrogenase region of Nox2 plays a key role in the interaction with p67phox.
abstract: p67(phox) 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 p67(phox) 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 p67(phox). For this purpose, we measured the binding of recombinant p67(phox) to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288-570). We found that: 1) p67(phox) 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 a (369)Cys-Gly-Cys(371) 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 (369)Cys 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 p67(phox), seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67(phox).
authors.affiliation: *The Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Israel; and Department of Biology and Physics, College of Science and Mathematics, Kennesaw State University, Kennesaw, Georgia, USA.
authors.forename: Iris
authors.initials: I
authors.lastname: Dahan
authors.affiliation: *The Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Israel; and Department of Biology and Physics, College of Science and Mathematics, Kennesaw State University, Kennesaw, Georgia, USA.
authors.forename: Susan M E
authors.initials: SM
authors.lastname: Smith
authors.affiliation: *The Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Israel; and Department of Biology and Physics, College of Science and Mathematics, Kennesaw State University, Kennesaw, Georgia, USA epick@post.tau.ac.il.
authors.forename: Edgar
authors.initials: E
authors.lastname: Pick
mesh_terms: D020816:Amino Acid Motifs; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D008562:Membrane Glycoproteins; D010455:Peptides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human
keywords: NADPH oxidase; chronic granulomatous disease; flavocytochrome b558; peptide arrays; superoxide