Protein structure encodes the ligand binding specificity in pheromone binding proteins.
The ligand specificities and binding affinities of three recombinant pheromone binding proteins (PBPs) of two saturniid moths (genus Antheraea) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [3H]-6E,11Z-hexadecadienyl acetate and [3H]-4E,9Z-tetradecadienyl acetate. The new binding assay, in which nonspecific adsorption to a plastic vessel is 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 [3H]-6E,11Z-16:Ac and [3H]-4E,9Z-14:Ac between 0.6 and 30 microM, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligands. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:Ac 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, displacement of [3H]-6E,11Z-16:Ac binding by other pheromone components or analogs showed a clear trend in relative binding affinity: 6E,11Z-16:Ac > 4E,9Z-14:Ac > 6E,11Z-16:Al approximately 16:Ac > 6E,11Z-16:OH > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguously demonstrate the specificity of the PBP-pheromone interaction. Moreover, this assay offers the potential for determining ligand specificities for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.
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Department of Chemistry, University at Stony Brook, New York 11794-3400, USA.
Prestwich
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D007301:Insect Hormones / Q000737:chemistry / Q000378:metabolism*; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
C094731:Aper-2 protein, Antheraea pernyi; D002352:Carrier Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009838:Oligodeoxyribonucleotides; C067715:APR-1 pheromone-binding protein, Antheraea pernyi