Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

Abstract

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 b 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 gp9l-phox encompassing residues S8TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E-5mSGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP'60 and G177GPPGGP'83). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp9l-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ40were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 ,LM and 230 ,uM, 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 b involves multiple binding sites along the C-terminal tails of both gp91-and p22-phox and other regions of gp9l-phox nearer to the N terminus.

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences

“…Finally, bovine gp91-phox contains a non-conserved substitution (Asn=Lys) at residue 430, eliminating a glycosylation motif, but has a second non-conserved substitution (Ala=Thr) at residue 471, resulting in the creation of a new consensus glycosylation motif for Asn469. Because this region of the protein is adjacent to several p47-phox binding domains [33,35,58] and consequently is oriented inside the cell (i.e., cytosolic), the glycosylation motif at Asn469, as was shown for the motif at Asn430 of the human protein [57], is probably not utilized as a target for glycosylation.…”

“…Potential N-linked glycosylation sites in the bovine amino acid sequence with the consensus sequence N-X-(S/T) are indicated by arrowheads. Other functional regions mapped to the human gp91-phox protein are underlined and include putative p47-phox binding sites (labeled [1][2][3][4] [33,35,57], potential flavin binding domains (labeled A and B) [29,44], and potential NADPH binding domains (labeled C-F) [28,29]. See text for further details.…”

“…However, because of the complex nature of this heterodimeric membrane protein, the specific functional and structural features of flavocytochrome b have not been fully characterized. Thus what little is known about the important functional and structural sites of these two proteins has come mainly from natural genetic mutations of these proteins [32], peptide mapping studies [33][34][35], and comparison of areas of conservation between the amino acid sequences obtained from other flavoproteins [28,29,36]. However, flavocytochrome b homologs have been sequenced from only a few species other than human.…”

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…7, B and D, was modified so that residues believed to contact p47 phox are shown in pink (Fig. 8) (33,(43)(44)(45). This view of gp91 phox shows residues 555 ESGPRGVHFIF 565 in pink (Phe 565 is red and represents the C terminus).…”

“…This view of gp91 phox shows residues 555 ESGPRGVHFIF 565 in pink (Phe 565 is red and represents the C terminus). This region of the molecule has been identified as a potential p47 phox contact site based on CGD analysis (46), synthetic peptide inhibition of oxidase activation (43,45) and translocation (44), and selection by phage display analysis of p47 phox affinity matrices (45). Such a placement also suggests that the C-terminal residues of gp91 phox , 566 NKENF 570 (not shown in the model), might lay over the cleft proposed to contain the NADPH binding site of gp91 phox and would allow Phe 570 to coordinate the isoalloxazine ring of the FAD as originally suggested by Taylor et al (47) from studies of similarity to the ferridoxin nucleotide reductases.…”