X-RAY CRYSTALLOGRAPHIC IDENTIFICATION OF A PROTEIN-BINDING SITE FOR BOTH ALL-TRANS-RETINOIC AND 9-CIS-RETINOIC ACID

The elucidation of how a protein-binding site might specifically recog nize both the all-trans and 9-cis isomers of retinoic acid is of parti cular interest because of the recently discovered binding specificitie s of the nuclear receptors for retinoic acid. Two families of nuclear receptors for retinoic acid have been described, which are designated RAR (for retinoic acid receptor) and RXR (for retinoid-X receptor). Th e RXR family of receptors is specific for 9-cis-retinoic acid, whereas the RAR-type receptor is activated by either 9-cis- or all-trans-reti noic acid. During the x-ray structure determination of a secreted epid idymal retinoic acid-binding protein, with and without retinoic acid, we observed an electron density for the bound all-trans-retinoic acid that indicates the protein-bound all-trans form of the vitamin/hormone adopts a horseshoe-like conformation that resembles the structure of the 9-cis isomer of the ligand. We detail here the experiments that in dicate the electron density is indeed due to all-trans-retinoic acid a nd that protein can also bind the 9-cis isomer. This observation and t he fact that the same protein also binds the synthetic retinoid ,8-tet ramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB), a retinoic acid analog that activates RAR but does not activate RXR, suggest that the mechanism by which this protein recognizes both 9-cis- and all-tr ans-retinoic acids may be analogous to the mechanism used by RAR. Thre e crystallographic structures of retinol-binding proteins have been de scribed. In each of these structures the retinol binds with the isopre ne tail fully extended. This report represents an x-ray crystallograph ic description of a protein-bound retinoid conformer that adopts a non extended conformation, and we believe this observation is relevant to the ligand specificities described for the retinoic acid receptors.