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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lefebvre
B
B
CJF-INSERM 92-03, Laboratoire de Biochimie Structurale, Faculté de Médecine de Lille, France.
Rachez
C
C
Formstecher
P
P
Lefebvre
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004330:Drosophila; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D000072482:Retinoic Acid Receptor alpha; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014212:Tretinoin / Q000378:metabolism*; D000097768:Retinoic Acid Receptor gamma
D008024:Ligands; D046911:Macromolecular Substances; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin; D000097768:Retinoic Acid Receptor gamma; C000607071:RARA protein, human; C082925:retinoic acid receptor beta