Background: Formyl peptide receptor 1 (FPR1) and FPR2 are highly homologous but bind fMet-Leu-Phe with very different affinities. Results: Asp-281 provides a negative charge that renders FPR2 more sensitive to the length and composition of formyl peptides than FPR1. Conclusion: Asp-281 is a major determinant for FPR2 binding. Significance: This work provides a structural basis for differential interaction between formyl peptides and their receptors.

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Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli chemottactic peptide fMet‐Leu‐Phe (fMLF). We investigated the structural requirements for FPR2 to interact with formyl peptides of different composition. In calcium flux assays, the N‐formyl group of these peptides is necessary for activation of both receptors while the composition of the C‐terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tripeptide (fMLF) and tetrapeptides (fMLFK, fMLFW), but not peptides with negatively charged side chains at the C‐terminus (e.g. fMLFE). In contrast, FPR1 does not discriminate against the C‐terminal negative charges. A CXCR4‐based homology model of FPR1 and FPR2 revealed that Asp2817.32 could be crucial for FPR2 interaction with certain formyl peptides as its negative charge might be repulsive with the terminal COO‐ group of fMLF and the negatively charged Glu in fMLFE. Asp2817.32 is a candidate for a stable interaction with the positively charged Lys in fMLFK. Site‐directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The resulting mutant, D2817.32G, showed a 10‐fold higher affinity for fMLFE and fMLF, but it reduced interaction with fMLFK compared with wild type FPR2. These results show that FPR1 and FPR2 use different structural determinants for interaction with formyl peptides.