Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.
Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution.
Chen
G
G
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Radominska-Pandya
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014212:Tretinoin / Q000737:chemistry / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D000596:Amino Acids; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D018168:Receptors, Retinoic Acid; C082927:retinoic acid binding protein I, cellular; D005973:Glutamine; D014316:Tritium; D014212:Tretinoin; D014364:Tryptophan; D001120:Arginine; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D008239:Lysine