Effects of ligand binding on the association properties and conformation in solution of retinoic acid receptors RXR and RAR

In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-t rans retinoic acid (RA), are involved in the regulation of several essentia l physiological processes. Their pleiotropic physiological effects are medi ated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuc lear hormone receptors. Hormone binding to the structurally conserved ligan d-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation. We report an extensive biophysical solution study of RAR alpha, RXR alpha L BDs and their corresponding RXR alpha /RAR alpha LED heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scatte ring (SAXS and SANS) and nb initio three-dimensional shape reconstruction a t low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthe rmore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXR alpha and RAR alpha LBDs in solution. The present study shows that in solution RAR alpha LED behaves as a monomer in both unliganded and liganded forms. It confirms the existence in soluti on of a ligand-induced conformational change towards a more compact form of the LED. It also confirms the stability of the predicted RXR alpha /RAR al pha LED heterodimers in solution. SAS measurements performed on three diffe rent types of RXR alpha /RAR alpha LED heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the exi stence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodi meric context. These results suggest that the subunits are structurally ind ependent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In sol ution unliganded RXR alpha LBD is observed as two species that are unambigu ously identified as homotetramers and homodimers. Molecular modelling combi ned with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXR alph a LBD bound to 9-cisRA is a homodimer regardless of the protein concentrati on. This study demonstrates the crucial role of ligands in the regulation o f homodimeric versus heterodimeric association state of RXR in the NR signa lling pathways.