Abstact

The retinoic acid-related orphan receptor alpha (RORalpha) is a potential drug target for cancer, inflammation, and metabolic diseases. Structure-guided ligand optimization can facilitate the development of selective RORalpha modulators. However, structural studies of the RORalpha ligand-binding domain (LBD) have been limited due to difficulties in purifying recombinant protein suitable for crystallographic analysis. Here, we engineered a chimeric RORalpha LBD C-terminally fused to the LXXLL motif of the coactivator RIP-140. This fusion improved the stability and solubility of the RORalpha LBD, enabling high-yield purification using the E. coli expression system. We determined the crystal structure of the chimeric RORalpha LBD in complex with cholesterol at 2.7 A resolution. The cholesterol-bound RORalpha LBD adopted an active conformation of helix 12 that accommodates coactivator binding. The coactivator peptide appears to stabilize the RORalpha LBD by shielding the hydrophobic surface of the AF-2 region. Although monomeric in solution, the RORalpha LBD-LXXLL fusion formed a dimer in the crystal lattice through extensive interactions involving the LXXLL motifs and alpha3 helices, which may represent a physiological homodimer. Fusion of a coactivator motif to the LBD is expected to facilitate structural studies of RORalpha and may be broadly applicable to other nuclear receptor LBDs for generating stable recombinant proteins.