STRUCTURAL DETERMINANTS OF THE LIGAND-BINDING SITE OF THE HUMAN RETINOIC ACID RECEPTOR-ALPHA

The ligand-dependent transactivating properties of retinoic acid receptors are controlled through a complex structure at the C-terminus of these proteins, commonly referred to as the hormone binding domain. This domain is involved not only in ligand recognition but also in protein-protein interactions such as homo- and heterodimerization processes. To identify more precisely regions of the human all-trans-retinoic acid receptor alpha (hRAR alpha) that are involved in Ligand binding, we constructed a series of deletion mutants of this molecule and overexpressed them in bacteria. We found that the C-terminal part of the D domain (amino acids 186-198) was necessary for ligand binding. The F domain and the 10 C-terminal amino acids of the E domain were dispensable for high-affinity binding of various natural and synthetic retinoids. A further deletion to position 403 resulted in a moderate decrease in affinity for all-trans-(ATRA) and 9-cis-retinoic acids, whereas the binding of two RAR alpha-specific ligands (Am80 and Am580) was abolished. In addition, hRAR alpha and the minimal hormone binding domain (amino acids 186-410) bound ATRA with a positive, cooperative mechanism. This behavior was not observed with CD367, a conformationally restricted synthetic retinoid. The positive cooperativity could be correlated with stable ATRA binding to RAR homodimers, whose formation was triggered by ligand. In the same conditions, only monomeric CD367-RAR alpha complexes were detected. These data indicate that ligand binding to hRAR alpha requires the presence of part of the D domain, whereas the C-terminal end of the E domain is involved in more subtle ligand recognition processes, They also clearly suggest that structurally distinct retinoids interact differently with the Ligand-binding site of this receptor.