Mutation of two residues converts the ligand-binding domain of RXRα into a uniform monomer without impairing the binding of retinoic acid and cofactors

Abstract

Retinoid X receptor (RXRα) is a nuclear receptor (NR) for retinoic acid (RA) and regulates various NR signaling pathways. Ligand-binding domain (LBD) of RXRα can bind with its ligand 9-cis-RA and cofactors, and mediate the forming of homodimer and homotetramer of RXRα and its heterodimer with other NRs, conferring RXRα the ability to play complicated roles in development and diseases. Due to the coexistence of monomer, dimer and tetramer, there are difficulties to study the structure and interaction of RXRα-LBD with its ligands and cofactors in solution and to distinguish the roles of different forms of RXRα in cell. Here, through analyzing available structures of RXRα-LBD, we selected two residues, D379 and L420, in the homodimer interface to design three mutants of RXRα-LBD. Recombinant proteins of the three mutants showed decreased proportions of dimer and tetramer but unchanged overall structure and binding affinities to 9-cis-RA, corepressor SMRT, and coactivator SRC2. Especially, the double-site mutant RXRα-LBDD379A−L420G existed as a uniform monomer. Furthermore, L420 was found to play a similar role in forming RXRα-LBD homodimer and its heterodimer with various NRs, while the role of D379 varies a lot, as it shows almost no interaction with RARα/β, LXRα/β, and THRα/β. This study provides a new insight into the mechanism for forming RXRα-LBD homodimer and its heterodimer with other NRs, and will facilitate the studies on the structure and interaction of RXRα-LBD with ligands, cofactors and drugs in solution, and the broad physiological functions of RXRα cooperating with various NRs in cell.