Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar

The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyocta decane, (+)-disparlure, as a sex pheromone, The (-) enantiomer of the phero mone is a strong behavioral antagonist. Specialized sensory hairs, sensilla e, on the antennae of male moths detect, the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) trans ports the odorant to the sensory neuron. We have expressed the two PBPs fou nd in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study phero mone binding under equilibrium conditions, we developed and validated a bin ding assay. We have addressed the two major problems with hydrophobic ligan ds in aqueous solution: (I) concentration-dependent adsorption of the ligan d on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate fur the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 ha s a higher affinity for the (-) enantiomer, while PBP2 has a higher affinit y for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polypl emus (Apol-3) bound the disparlure enantiomers more weakly than either of t he L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers . We have observed extensive aggregation of both L. dispar PBPs and an incr ease in pheromone binding at high (>2 mu M) PBP concentrations. We present a model of disparlure binding to the two PBPs.