MAPPING THE AUXIN-BINDING SITE OF AUXIN-BINDING PROTEIN-1

Auxin-binding protein 1 (ABP1) is a putative receptor for the class of plant growth hormones designated auxins of which indole-3-acetic acid (IAA) is the predominant endogenous member. ABP1 is a homodimeric gly coprotein consisting of subunits of 163 amino acid residues. We have p erformed a structural study of ABP1 that has localized a region along its primary sequence that is involved in hormone binding, We have used the photoaffinity labeling agent, 5-[7-H-3]azidoindole-3-acetic acid (5-[H-3]N(3)IAA), an active auxin analog, to covalently label residues that are within, or near, the auxin-binding site. Photolabeled ABP1 w as digested to completion with trypsin, and the resulting peptides wer e purified by reverse phase high performance liquid chromatography. Wh en 5-[H-3]N(3)IAA was used at a concentration of 0.5 mu M (one order o f magnitude below the K-d for 5-N(3)IAA) only one peptide was labeled at a high specific activity, Labeling was blocked by the presence of 5 0 mu M IAA, indicating that the interaction is specific, Sequence anal ysis determined that this tryptic fragment was derived from ILe(130) t o Leu(145) of ABP1. We suggest that residue Asp(134) is the specific t arget of the photolabeling and is within 1.48 Angstrom of the postulat ed hydrophobic platform of the auxin-binding site. We propose that Trp (136) may serve as this hydrophobic platform in the binding site for t he aromatic rings of auxins.