Evidence for protonation in the human neutrophil formyl peptide receptor binding pocket.

We have studied the interaction of a family of fluorescent formyl peptides with their receptor using spectrofluorometric and flow cytometric methods. The peptides contained four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys-fluorescein), or six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys- fluorescein) amino acids. As observed in earlier studies, the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, while the hexapeptide was not. While the degree of quenching of the bound tetrapeptide was largely unchanged, the quenching of the bound pentapeptide decreased with increasing pH over the range of pH 6.5-9.0. Ligand binding studies have shown that the mole fraction of tetrapeptide or pentapeptide bound in kinetic analysis markedly decreased with increasing pH as a consequence of increasing ligand dissociation rate constant. The dependence of the binding parameters for the hexapeptide on pH was much less pronounced. Over a pH range from pH 7.3 to 9.0, the hexapeptide showed little change in binding affinity, while the tetrapeptide and pentapeptide increased in Kd approximately 2.0- and 2.5-fold, respectively. These results indicate that the formyl peptide receptor binding pocket contains at least two microenvironments. The pH sensitivity of the pentapeptide quenching is consistent with a protonating environment, while the pH-independent quenching of the tetrapeptide may reflect aromatic stacking or a hydrophobic microenvironment. The pH-dependent ligand dissociation also suggests that the protonation in the pocket stabilizes ligand binding, which may indicate an alteration in the binding pocket structure. Protonation or hydrogen bonding of the pentapeptide may lead to even further stabilization of that ligand.