Critical role of the H6-H7 loop in the conformational adaptation of all-trans retinoic acid and synthetic retinoids within the ligand-binding site of RARalpha

Abstract

The pleiotropic effects of the natural and synthetic retinoids are mediated by the activation of the two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). At the molecular level, these events begin with the specific ligand recognition by a nuclear receptor subtype. The adaptation of ligands to the receptor binding site leads to an optimal number of interactions for binding and selectivity which justifies elucidation of the structural requirements of the ligand binding pocket. To explore the contribution of H6-H7 loop folding in the ligand-induced conformational changes explained by the mouse-trap model, four RAR mutants were constructed. Ligand binding and transactivation studies revealed that three residues from the H6-H7 loop (Gly 301 , Phe 302 and Gly 303) are critical for the conformational adaptation of both synthetic agonists and antagonists. Model building and analysis of both RAR -ATRA and RAR -CD367 complexes demonstrate that accommodation of CD367 results in a less tight contact of the saturated ring of this ligand with the amino acid side chains of the receptor ligand-binding pocket compared with that of ATRA. According to the flexibility of the agonists tested (ATRA>TTNPB=Am580> CD367), we observed a decrease in binding that was dependent on ligand structure rigidity. In contrast, the binding and transactivating activities of the L266A mutant confirmed the structural constraints imposed by synthetic ligands on binding affinity for the receptor and revealed that subtle local rearrangements induced by specific conformational adaptation changes result in different binding affinities. Our results illustrate the dynamic nature of the interaction between RAR and its ligands and demonstrate the critical role of the H6-H7 loop in the binding of both synthetic retinoid agonists and antagonists.

New computational method for prediction of interacting protein loop regions

“…In many proteins, loop regions are critical to the overall function of the protein. Loop regions have been shown to be involved in ligand binding,1–14 enzymatic catalysis,15–20 and protein–protein recognition 21–23. In general, loop regions are more flexible than α‐helices and β‐sheets and often undergo conformational change upon ligand or protein binding.…”

Synthetic Retinoids and Their Nuclear Receptors

“…Thus, both the trans-RA (1) and 9-cis-RA (6) carboxylates were anchored in the RARγ LBP by their salt bridges with the H5 arginine-278 and were hydrogen-bonded to other LBP residues and a water molecule [211,212]. Mutational studies on the RARα LBD indicated that agonists induced different conformations in three 6-7 loop residues (glycine-301, phenylalanine-302, and glycine-303) compared to antagonists [228], thereby suggesting that this region also has an important role in transactivation. In the holo-RARγ-trans-RA (1) complex, the ligand occupied 66% (291 Å 3 ) of the 441-Å 3 holo-LBP [204], and its β-ionone ring (hydrophobic terminus) interacted with lipophilic side-chains, which pointed into the pocket [211].…”

Predicting flexible loop regions that interact with ligands: The challenge of accurate scoring

“…Although the configuration of loop regions is frequently incorrect in homology models, these structural elements are often critical to the biological function of many proteins 4–25. Loop regions have been shown to be involved in processes ranging from protein–protein interactions to ligand binding.…”