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 Tm of 58.7 °C and an unfolding enthalpy (ΔH) 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 (ΔCp) of 15 kJ/(mol·K) determined by measurements at different pH values, the free energy of unfolding (ΔG) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 °C, and increased the ΔG of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. The increase in ΔG 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 ΔG 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.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- RXRα LBD
- Retinoid X Receptor-alpha Ligand Binding Domain
- LBP
- ligand binding pocket
- 9cRA
- 9-cis retinoic acid
- GRIP-1
- glucocorticoid receptor interacting protein-1
- RXRα LBD:UAB30
- RXRα LBD bound to UAB30
- RXRα LBD:UAB30:GRIP-1
- RXRα LBD bound to UAB30 and GRIP-1
- RXRα LBD:UAB110
- RXRα LBD bound to UAB110; RXRα
- LBD:UAB110:GRIP-1
- RXRα LBD bound to UAB110 and GRIP-1; RXRα
- LBD:UAB111
- RXRα LBD bound to UAB111; RXRα
- LBD:UAB111:GRIP-1
- RXRα LBD bound to UAB111 and GRIP-1
- SRC
- steroid receptor coactivator
- DSC
- differential scanning calorimetry
- v
- scan or heating rate
- Tm
- thermal unfolding temperature
- ΔHv
- van’t Hoff enthalpy of unfolding
- ΔHc
- calorimetric enthalpy
- ΔG
- Gibbs free energy of unfolding
- ΔS
- entropy of unfolding
- ΔCpu
- unfolding heat capacity change
- Ku
- unfolding equilibrium constant
- DSF
- differential scanning fluorimetry
- F350/F330
- ratio of fluorescence intensity at 350 nm divided by fluorescence intensity at 330 nm
- λex
- excitation wavelength for fluorescence measurements
- CD
- Circular Dichroism
- ITC
- isothermal titration calorimetry
- ΔHa
- binding enthalpy
- ΔCpa
- binding heat capacity change
- Ka
- ligand binding constant
- Kd
- ligand dissociation constant
- ΔCpa
- heat capacity change upon binding
- HDX-MS
- hydrogen-deuterium exchange mass spectrometry
- N
- native folded protein
- U
- reversibly unfolded protein
- D
- irreversibly denatured protein
- I
- partially unfolded intermediate
- R
- rexinoid
- P
- coactivator peptide GRIP-1