Stability of the Retinoid X Receptor-alpha Homodimer in the Presence and Absence of Rexinoid and Coactivator Peptide

ABSTRACT Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-alpha ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo -RXRα LBD homodimer displayed a single thermal unfolding transition with a T m of 58.7 °C and an unfolding enthalpy (Δ H ) of 673 kJ/mol (12.5 J/g), much lower than average value (35 J/g) of small globular proteins. Using a heat capacity change (Δ C p ) of 15 kJ/(mol·K) determined by measurements at different pH values, the free energy of unfolding (Δ G ) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo -homodimer increased T m by 5 to 9 °C, and increased the Δ G of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant ( K d ) of the rexinoids. The increase in Δ G was the result of a more favorable entropic change due to interactions between the rexinoid and hydrophobic residues in the binding pocket, with the larger increases caused by rexinoids containing larger hydrophobic end groups. GRIP-1 binding to holo -homodimers containing rexinoid resulted in additional increases in Δ G of 14 kJ/mol, a value same for all three rexinoids. Binding of rexinoid and GRIP-1 resulted in a combined 50% increase in unfolding enthalpy, consistent with reduced structural fluidity and more compact folding observed in other published structural studies. Thermodynamic analysis thus provided a quantitative evaluation of the interactions between RXR and its agonist and coactivator.