Interaction of human neutrophil flavocytochrome b with cytosolic proteins: transferred-NOESY NMR studies of a gp91phox C-terminal peptide bound to p47phox

Abstract

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.

Selective Role for β-Protein Kinase C in Signaling for O⨪2 Generation but Not Degranulation or Adherence in Differentiated HL60 Cells

“…phox is required for a conformational change in p47 phox , which releases binding of p47 phox to itself and to p40 phox (43), and allows translocation and binding of p47 phox to membrane-associated cytochrome b 558 (43)(44)(45)(46). In the present study, fMet-Leu-Phe and PMA triggered phosphorylation and translocation of p47…”

Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

“…The C-terminal domain of gp91 phox shows homology to the ferridoxin nucleotide reductase family of oxidoreductases (22), and models of this region have been generated by structure-based alignment programs (14,23). The ability of this domain to bind redox cofactors (NADPH and FAD) has been demonstrated by biochemical labeling experiments (22,24), whereas a variety of methods have been used to identify residues in the gp91 phox C-terminal domain that are critical for assembly of the NADPH oxidase complex (17,18,25,26). Current models localize the gp91 phox C-terminal domain to the cytoplasmic aspect of the plasma or phagosomal membrane (4,12,27).…”

Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system

“…1 This induces a conformational change in p47 phox , 9 which reveals binding sites for the flavocytochrome subunits. [10][11][12][13][14] Gene expression of gp91 phox , p47 phox , and p67 phox is largely restricted to mature phagocytes. Genetic defects in any 1 of the 4 essential phox components, gp91 phox , p22 phox , p47 phox , or p67 phox , result in chronic granulomatous disease.…”