Interaction of human neutrophil flavocytochrome b with cytosolic proteins: transferred-NOESY NMR studies of a gp91phox C-terminal peptide bound to p47phox.
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.
Adams
E R
ER
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT59717, USA.
Dratz
E A
EA
Gizachew
D
D
Deleo
F R
FR
Yu
L
L
Volpp
B D
BD
Vlases
M
M
Jesaitis
A J
AJ
Quinn
M T
MT
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000097:blood / Q000737:chemistry; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012694:Serine
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D012694:Serine; D010649:Phenylalanine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1