Direct Photoaffinity Labeling of Cellular Retinoic Acid-Binding Protein I (CRABP-I) with all-trans-Retinoic Acid: Identifcation of Amino Acids in the Ligand Binding Site

摘要

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 o fCRABP-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. Photolableled 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 o fspecific amino acids in the PA-binding site of CRABPs by photoaffinity lbeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution.