Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ

PLoS One. 2017 Jan 26;12(1):e0171043. doi: 10.1371/journal.pone.0171043. eCollection 2017.

Abstract

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

MeSH terms

  • Allosteric Regulation / physiology*
  • Humans
  • Ligands
  • Molecular Dynamics Simulation
  • Phosphorylation
  • Protein Binding
  • Receptors, Retinoic Acid / metabolism*
  • Tretinoin / metabolism*

Substances

  • Ligands
  • Receptors, Retinoic Acid
  • retinoic acid receptor gamma
  • Tretinoin

Grant support

This work was supported by funds from the Centre National de Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg, Agence Nationale pour la Recherche (ANR-09-BLAN-0127-01) and the Institut National du Cancer (INCa-PL09-194), Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions, the French Infrastructure for Integrated Structural Biology (FRISBI) (Grant ANR-10-INSB-05-01). This study was also supported by the grant ANR-10-MABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.