Thermodynamic characterization of the interaction between CAR-RXR and SRC-1 peptide by isothermal titration calorimetry.

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.