INTERACTION OF HUMAN NEUTROPHIL FLAVOCYTOCHROME-B WITH CYTOSOLIC PROTEINS - TRANSFERRED-NOESY NMR-STUDIES OF A GP91(PHOX) C-TERMINAL PEPTIDE BOUND TO P47(PHOX)

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochr ome b via multiple binding regions, including a site in the C-terminus of gp91(Phox). To investigate this binding site further, we studied t he three-dimensional structure of a gp91(Phox) C-terminal peptide ((55 1)SNSESGPRGVHFIFNKEN(568)) bound to p47(Phox) using transferred nuclea r Overhauser effect spectroscopy (Tr-NOESY) NMR, Using MARDIGRAS analy sis and simulated annealing, five similar sets of structures of the p4 7(phox)-bound peptide were obtained, all containing an extended open b end from Sers to Phe(14) (corresponding to gp91(Phox) residues 555-564 ). The ends of the peptide were poorly defined, however, suggesting th ey were more flexible. Therefore further refinement was performed on t he Ser(5)-Phe(14) 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 conforma tions. 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. T hus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 55 5 and 564, with immobilization of all of the amino acid side chains in the (558)RGVHFIF(564) region except for His(561).