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 (RXRa LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXR alpha LBD homodimer displayed a single thermal unfolding transition with a T-m 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 C-p) of 15 kJ/(mol 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 T-m by 5 to 9 degrees C and increased the.G of the native homodimer by 12 to 20 kJ/mol at 37 degrees C, consistent with the nanomolar dissociation constant (K-d) of the rexinoids. GRIP-1 binding to holo-homodimers containing rexinoid resulted in additional increases in Delta G of 14 kJ/mol, a value that was the 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. The complexes of UAB110 and UAB111 are each more stable than the UAB30 complex by 8 kJ/mol due to enhanced hydrophobic interactions in the binding pocket because of their larger end groups. This increase in thermodynamic stability positively correlates with their improved RXR activation potency. Thermodynamic measurements are thus valuable in predicting agonist potency.