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 tra nslocates to the plasma membrane and binds to flavocytochrome b, and b inding domains for p47-phox have been identified on the C-terminal tai ls of both flavocytochrome 6 subunits. In the present report, we furth er examine the interaction of these two oxidase components by using ra ndom-sequence peptide phage display library analysis. Screening p47-ph ox with the peptide libraries identified five potential sites of inter action with flavocytochrome b, including three previously reported reg ions of interaction and two additional regions of interaction of p47-p hox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phos encompassin g residues S(86)TRVRRQL(93) and to a domain near the cytosolic C-termi nal tail of gp91-phos encompassing residues F(150)EWFADLL(457). The ma pping also confirmed a previously reported binding domain on gp91-phox (E(554)SGPRGVHFIF(564)) and putative Src homology 3 domain binding si tes on p22-phox (P(156)PRPP(160) and G(177)GPPGGP(183)). To demonstrat e 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 ab ility to inhibit NADPH oxidase activity. These peptides had EC(50) val ues of 1 mu M and 230 mu M, respectively, and inhibited activation whe n added prior to assembly but did not affect activity of the preassemb led oxidase. Our data demonstrate the Usefulness of phage display libr ary analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to f lavocytochrome 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.