Kinetic studies of pheromone-binding proteins (LdisPBP1 and LdisPBP2) from spongy moth Lymantria dispar with natural ligands and their analogues: Enantiomer distinction at external and internal binding sites

Olfaction plays an important role in the reproduction of the spongy moth, Lymantria dispar, a major defoliator of deciduous trees. Detection of the attractant sex pheromone, (7R, 8S)-epoxy-2-methyloctadecane, (+)-disparlure, aids males in the location of mates by triggering upwind flight behavior. The antipode, (-)-disparlure, inhibits upwind flight behavior. The sensory hairs on the antennae of the male moth are innervated by the dendrite of an olfactory neuron, which is immersed in sensory lymph, a solution with pheromone-binding proteins (PBPs). These proteins bind pheromone molecules selectively and are necessary for proper olfactory functioning. Two PBPs from spongy moth are known: LdisPBP1 and LdisPBP2. The two proteins exhibit differences in their binding affinities towards various natural and unnatural ligands at their internal binding sites. Ligands are bound by PBPs in a two-step process: ligand capture results in binding to an external site, which is followed by movement of the ligand to the internal site. In this study, we investigate the differences in association and dissociation rates of the two proteins with (+)-disparlure, its enantiomer, (-)-disparlure, and some analogues with heteroatom substitutions of oxygen or sulfur at either carbon-5 or carbon-10, using stopped-flow kinetics. We used a fluorescent reporter (N-phenyl-1-naphthylamine, NPN), which competes with the ligands of interest and reports on external site binding in PBPs. The results are discussed in the context of kinetic selectivity of PBPs towards natural disparlure and synthetic analogue ligands, at their external binding sites, where enantiomer distinction has not been detected previously. Ligand docking simulations at two external sites of A and B forms and at one internal site of the B forms of the two proteins were used to interpret the enantioselectivity in the association and dissociation of the ligands. This paper also discusses the enantiomer selectivity and the molecular role of PBPs in the desorption from hydrophobic surfaces and partition between lipid vesicles and aqueous solutions of the pheromone enantiomers and their heteroatom-substituted analogues. Results are interpreted in terms of the contribution of ligand association and dissociation kinetics to the enantiomer selectivity of the spongy moth PBPs.