Structure-function relationship in the interaction of mastoparan analogs with neutrophil NADPH oxidase

Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown t o block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxid ase activation was now demonstrated in the semirecombinant NADPH oxidase sy stem. The structural basis of the inhibitory effect of MP on oxidase assemb ly was explored employing a variety of truncated and specifically substitut ed synthetic peptide analogs. The data indicated that an cu helical fold, p ositive net charge, hydrophobicity and amphiphilicity were essential for th e inhibitory potency and that peptide analogs below eleven residues were in active. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rad in conjunction with immobilized MP or soluble I -125-tyr-MP, respectively. The data implicated p67-phox as the main MP-bind ing component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-pho x and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a l ow affinity towards MP. (C) 2001 Elsevier Science Inc. Ah rights reserved.