Identification of a Ligand Binding Site in the Human Neutrophil Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity Label and Mass Spectrometry

Abstract

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...

Characterization of the Binding Site on the Formyl Peptide Receptor Using Three Receptor Mutants and Analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

“…In addition, our previous cross-linking data suggest that the leucine side chain of fMLF is probably located close to FPR 93 VRK 95 , which is at the COOH terminus of helix II (14). Therefore, the most likely positioning of f MLF in the binding pocket of FPR is as follows.…”

“…We previously developed a model for fMLF binding to FPR (28) based on: 1) the structural similarities between fMLF and all-trans-retinal and the fact that mutation of Phe-291 (VII-11), which is analogous to the lysine that forms the Shiff base with retinal in rhodopsin, markedly affected binding (13); 2) the fact that several additional residues that affect fMLF binding are also known to be important in retinal binding (13); and 3) a photoaffinity label of fMLF with a photoaffinity label in the leucine position cross-linked to helix II near the extracellular space (14).…”

“…Ten residues which affect fMLF binding have been mapped to transmembrane domains II-VII (13), including residues Leu-78 (II-17, helix II, residue 17 of 26 transmembrane-spanning residues in the nomenclature used throughout this text), Asp-106 (III-8), Leu-109 (III-11), Thr-157 (IV-18), Arg-201 (V-2), Ile-204 (V-5), Arg-205 (V-6), Trp-254 (VI-16), Tyr-257 (VI-19), and Phe-291 (VII-11). In addition, photo-cross-linking data suggest that the leucine side chain of fMLF is probably located close to FPR 93 VRK 95 , which is at the COOH terminus of helix II (14).…”

“…We have previously shown that the formyl peptide binding site maps to several membrane-spanning regions (13)(14). Ten residues which affect fMLF binding have been mapped to transmembrane domains II-VII (13), including residues Leu-78 (II-17, helix II, residue 17 of 26 transmembrane-spanning residues in the nomenclature used throughout this text), Asp-106 (III-8), Leu-109 (III-11), Thr-157 (IV-18), Arg-201 (V-2), Ile-204 (V-5), Arg-205 (V-6), Trp-254 (VI-16), Tyr-257 (VI-19), and Phe-291 (VII-11).…”

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

“…By integrating MS with PAL, researchers have not only identified binding partners for a particular ligand or receptor of interest, but also localized and characterized the sites of interaction at a molecular level [19][20][21][22][23][24][25] with far greater efficiency and rapidity than could be achieved with more traditional biophysical techniques. At the protein level, comparing the mass of photolabeled protein-protein ligand-receptor complexes with that of unlabeled material can be used to generate information on the stoichiometry of interactions [18].…”