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Pheromone-binding proteins (PBPs) are small, water-soluble proteins found in the lymph of pheromone-sensing hairs. PBPs are essential in modulating pheromone partitioning in the lymph and at pheromone receptors of olfactory sensory neurons. The function of a PBP is associated with its ability to structurally convert between two conformations. Although mechanistic details remain unclear, it has been proposed that the structural transition between these forms is affected by two factors: pH and the presence or absence of ligand. To better understand the PBP conformational transition, the structure of the gypsy moth ( Lymantria dispar ) LdisPBP1 was elucidated at pH 4.5 and 35 °C using nuclear magnetic resonance spectroscopy. In addition, the effects of sample pH and binding of the species' pheromone, (+)-disparlure, (7 R ,8 S )-epoxy-2-methyloctadecane, and its enantiomer were monitored via 15 N HSQC spectroscopy. LdisPBP1 in acidic conditions has seven helices, with its C-terminal residues forming the seventh helix within the pheromone-binding pocket and its N-terminal residues disordered. Under conditions where this conformation is made favorable, free LdisPBP1 would have limited ligand binding capacity due to the seventh helix occupying the internal binding pocket. Our findings suggest that even in the presence of 4-fold ligand at acidic pH, LdisPBP1 is only ∼60% in its pheromone-bound form. Furthermore, evidence of a different LdisPBP1 form is seen at higher pH, with the transition pH between 5.6 and 6.0. This suggests that LdisPBP1 at neutral pH exists as a mixture of at least two conformations. These findings have implications concerning the PBP ligand binding and release mechanism.

The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants., (Copyright © 2015 Elsevier Inc. All rights reserved.)