Binding of Fluorinated Phenylalanine α-Factor Analogues to Ste2p: Evidence for a Cation−π Binding Interaction between a Peptide Ligand and Its Cognate G Protein-Coupled Receptor

摘要

Ste2p, a G protein-coupled receptor (GPCR), binds R-factor, WHWLQLKPGQPMY, antridecapeptide pheromone secreted by yeast cells. Upon R-factor binding, Ste2p undergoes conformationalnchanges activating a signal transduction system through its associated heterotrimeric G protein leading to thenarrest of cell growth in the G1 phase to prepare cells for mating. Previous studies have indicated that Tyr atnposition 13 of R-factor interacts with Arg58 on transmembrane one (TM1) of Ste2p. This observationnprompted this investigation to determine whether a cation-π type of interaction occurred between thesenresidues. Tyrosine at position 13 of R-factor was systematically substituted with analogous amino acids withnvarying cation-π binding energies using solid-phase peptide synthesis, and these analogues were modified bynderivatization of their Lysn7nresidue with the fluorescent group 7-nitrobenz-2-oxa-1,3-diazole (NBD) to servenas a useful probe for binding determination. Saturation binding of these peptides to Ste2p was assayed usingnwhole yeast cells and a flow cytometer. In parallel the biological activities of the peptides were determinednusing a growth arrest assay. The data provide evidence for the presence of a cation-π interaction betweennArg58 of Ste2p and Tyrn13nof R-factor.