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

J Mol Biol. 2001 Mar 23;307(2):557-76. doi: 10.1006/jmbi.2000.4409.


In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-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 LBDs and their corresponding RXR alpha/RAR alpha LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore 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 LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXR alpha/RAR alpha LBD heterodimers in solution. SAS measurements performed on three different types of RXR alpha/RAR alpha LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In solution unliganded RXR alpha LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined 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 alpha LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoproteins / metabolism
  • Binding Sites
  • Computer Simulation
  • Crystallography / methods
  • Dimerization
  • Humans
  • Ligands
  • Models, Molecular
  • Mutation
  • Neutrons
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Receptors, Retinoic Acid / chemistry
  • Receptors, Retinoic Acid / genetics
  • Receptors, Retinoic Acid / metabolism*
  • Retinoid X Receptors
  • Scattering, Radiation
  • Solutions
  • Stereoisomerism
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcriptional Activation
  • Tretinoin / metabolism*
  • Ultracentrifugation
  • X-Rays


  • Apoproteins
  • Ligands
  • Receptors, Retinoic Acid
  • Retinoid X Receptors
  • Solutions
  • Transcription Factors
  • Tretinoin