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 glycoprotein consisting of subunits of 163 amino acid residues. We have performed 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-[7-3H]azidoindole-3-acetic acid (5-[3H]N3IAA), an active auxin analog, to covalently label residues that are within, or near, the auxin-binding site. Photolabeled ABP1 was digested to completion with trypsin, and the resulting peptides were purified by reverse phase high performance liquid chromatography. When 5-[3H]N3IAA was used at a concentration of 0.5 microM (one order of magnitude below the Kd for 5-N3IAA) only one peptide was labeled at a high specific activity. Labeling was blocked by the presence of 50 microM IAA, indicating that the interaction is specific. Sequence analysis determined that this tryptic fragment was derived from Ile130 to Leu145 of ABP1. We suggest that residue Asp134 is the specific target of the photolabeling and is within 1.48 A of the postulated hydrophobic platform of the auxin-binding site. We propose that Trp136 may serve as this hydrophobic platform in the binding site for the aromatic rings of auxins.
Brown
J C
JC
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Jones
A M
AM
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007210:Indoleacetic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D010937:Plant Growth Regulators; D010940:Plant Proteins; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C046131:auxin receptor, plant