The phagocyte chemotactic receptors, including the formyl peptide receptor (FPR), 1 the lipoxin A 4 receptor, the C5a receptor, the platelet-activating factor receptor, and the interleukin-8 receptor are involved in inflammation and are all members of the G protein-coupled receptor (GPCR) superfamily. Among the most studied in this inflammatory receptor family is neutrophil FPR (1). FPR binds N-formyl peptides, such as formyl-Met-Leu-Phe (fMLF), with nanomolar affinity (2). Such N-formyl peptides are indicators of the presence of bacteria (3) or damage to host cell mitochondria (4,5). Binding of N-formyl peptides to FPR thus provides phagocytes with signals for infection or injury and results in activation of chemotaxis and other host defensive processes including lysosomal enzyme secretion, stimulation of production of inflammatory mediators, and generation of superoxide.The effects of amino acid substitutions and modifications of fMLF peptides on binding to FPR and activation have been studied extensively (6 -8). The formyl group, the methionine at position 1, and phenylalanine at position 3 have been shown to be necessary for high affinity binding. Decarboxylation of the C-terminal phenylalanine markedly reduces activity, but esters or amides of this residue or peptides with C-terminal amino acid additions exhibit similar activity to the tripeptide with the free acid. None of the fMLF functional groups have been shown to be absolutely essential for activity but rather they appear to individually contribute to the overall free energy of binding.Chemical and photoaffinity cross-linking of fMLF analogs to FPR has been achieved by a number of groups (9 -12). However, none of these studies have identified the site of labeling. The residue in the second position of N-formylated peptides appears to be the most tolerant of modification (6, 13) and both of the flanking residues are critical for high affinity binding (14); so the second residue was chosen to accommodate the photoreactive amino acid benzoylphenylalanine (Bpa). Bpa is chemically stable in the absence of photoexcitation, can be directly introduced into peptide ligands by solid phase peptide synthesis, and has been photocross-linked into several peptide receptors (15). Here we report that a fluorescent photoaffinity analog of fMLF, formyl-Met-p-benzoyl-L-phenylalanine-PheTyr-Lys-⑀-N-fluorescein (fMBpaFYK-fl), efficiently photocrosslinks to FPR residues 83-85. Derivatization of these residues in FPR by Bpa supports recent site-directed mutagenesis stud-* This work was supported in part by National Science Foundation EPSCoR Grant RII-891879 (to E. A. D.), a Grant from the Pittsburgh Supercomputing Centers through the National Institutes of Health resource Grant 2p41RR06009, a grant from the Rocky Mountain Chapter of the Arthritis Foundation and the Harmon Foundation (to J. S. M.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (to H. M. M.), and Public Health Service Grants 1RO1A40108-01 and RO122735 (to A. J. J.). The costs of publicati...

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 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 conformations. 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. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.

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