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 odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis 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 to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis 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 pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.
Briand
L
L
Biochimie et Structure des Protéines, Unité de recherches INRA 477, Domaine de Vilvert, Jouy-en-Josas, France.
Nespoulous
C
C
Huet
J C
JC
Takahashi
M
M
Pernollet
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees; D001665:Binding Sites; D012831:Bombyx; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins; D008024:Ligands; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010675:Pheromones / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
C108516:ASP2 protein, Apis mellifera; D002352:Carrier Proteins; D004220:Disulfides; D019476:Insect Proteins; D008024:Ligands; D010675:Pheromones; D011994:Recombinant Proteins; D014357:Trypsin