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, respecti vely) are transport proteins for all-trans-retinoic acid (RA), an active me tabolite of vitamin A (retinol), and have been reported to be directly invo lved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,-12-H-3]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [H-3]RA was light- and concentration-dependent and was protected by unlabeled RA and v arious retinoids, indicating that the labeling was directed to the RA-bindi ng site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to y ield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified fi ve modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg 29, Lys38, and Trp109. All five amino acids are located within one side of the ''barrel" structure in the area indicated by the reported crystal struc ture as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity lab eling. These results provide significant information about the ligand bindi ng site of the CRABP-I molecule in solution.