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

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-alpha ligand binding domain (RXR LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXR LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 {degrees}C and an unfolding enthalpy ({Delta}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 ({Delta}Cp) of 15 kJ/(mol{middle dot}K) determined by measurements at different pH values, the free energy of unfolding ({Delta}G) of the native state was 33 kJ/mol at 37 {degrees}C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 {degrees}C, and increased the {Delta}G of the native homodimer by 12 to 20 kJ/mol at 37 {degrees}C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. The increase in {Delta}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 {Delta}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.