PROTEIN-STRUCTURE ENCODES THE LIGAND-BINDING SPECIFICITY IN PHEROMONEBINDING-PROTEINS

The ligand specificities and binding affinities of three recombinant p heromone binding proteins (PBPs) of two satumiid moths (genus Antherae a) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [H-3]-6E,11Z- hexadecadienyl acetate and [H-3]-4E,9Z-tetradecadienyl acetate. The ne w binding assay, in which nonspecific adsorption to a plastic vessel i s suppressed by presaturation of the surface with a 1-alkanol, allows measurement of dissociation constants (KD) for lipophilic ligands for their carrier proteins. The three PBPs showed KD values for [H-3]-6E,1 1Z-16:Ac and [H-3]-4E,9Z-14:Ac between 0.6 and 30 mu M, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligand s. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:A c than for 4E,9Z-14:Ac, while Aper-2 showed a 3.5-fold preference for binding the shorter chain compound. In addition, for the Apol-3 PBP, d isplacement of [H-3]-6E,11Z-16:Ac binding by other pheromone component s or analogs showed a clear trend in relative binding affinity: 6E,11Z -16:Ac > 4E,9Z-14:Ac > 6E,11Z-16,Al approximate to 16:Ac > 6E,11Z-16:O H > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguou sly demonstrate the specificity of the PBP-pheromone interaction. More over, this assay offers the potential for determining ligand specifici ties for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.