Chen G, Radominska-Pandya A
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Biochemistry. 2000 Oct 17;39(41):12568-74. doi: 10.1021/bi000321n.
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.
细胞视黄酸结合蛋白I和II（分别为CRABP-I和CRABP-II）是全反式视黄酸（RA）的转运蛋白，RA是维生素A（视黄醇）的一种活性代谢产物，据报道它们直接参与RA的代谢。在本研究中，使用[11,12-(3)H]RA进行直接光亲和标记，以鉴定构成CRABP-I配体结合位点的氨基酸。用[(3)H]RA对CRABP-I进行光亲和标记具有光依赖性和浓度依赖性，并受到未标记的RA和各种类视黄醇的保护，这表明该标记是针对RA结合位点的。光标记的CRABP-I用内肽酶Lys-C水解以产生放射性肽，这些肽通过反相高效液相色谱分离，用于通过埃德曼降解肽测序进行分析。该方法从五个单独的高效液相色谱馏分中鉴定出五个修饰的氨基酸：Trp7、Lys20、Arg29、Lys38和Trp109。所有这五个氨基酸都位于“桶状”结构的一侧，该区域在报道的晶体结构中被指定为配体结合位点。这是首次通过光亲和标记直接鉴定CRABPs的RA结合位点中的特定氨基酸。这些结果提供了有关溶液中CRABP-I分子配体结合位点的重要信息。
