MAPPING SITES OF INTERACTION OF P47-PHOX AND FLAVOCYTOCHROME-B WITH RANDOM-SEQUENCE PEPTIDE PHAGE DISPLAY LIBRARIES

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome 6 subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phos encompassing residues S(86)TRVRRQL(93) and to a domain near the cytosolic C-terminal tail of gp91-phos encompassing residues F(150)EWFADLL(457). The mapping also confirmed a previously reported binding domain on gp91-phox (E(554)SGPRGVHFIF(564)) and putative Src homology 3 domain binding sites on p22-phox (P(156)PRPP(160) and G(177)GPPGGP(183)). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F(77)LRGSSACCSTRVRRQL(93) and E(451)WFADLLQLLESQ(463) were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC(50) values of 1 mu M and 230 mu M, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the Usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome 6 involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.