Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)

In insects, the transport of airborne, hydrophobic odorant:; and pheromones through the sensillum lymph is generally thought to be accomplished by odo rant-binding proteins (OBPs). We report the structural and functional prope rties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds wen: assigned after classic trypsinol ysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found t o be identical to that of Bombyx mori OBP: suggesting that this pattern occ urs commonly throughout the highly divergent insect OBPs. CD measurements r evealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration an alysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pherom ones by ASP2. Recombinant ASP2 bound all tested molecules, except beta -ion one, which could not interact with it at all. The affinity constants of ASP 2 for these ligands, determined at neutral pH by isothermal titration calor imetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probabl y one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlati on with monomerization and a local conformational change supported by CD ex periments.