Chebaro Yassmine, Sirigu Serena, Amal Ismail, Lutzing Régis, Stote Roland H, Rochette-Egly Cécile, Rochel Natacha, Dejaegere Annick
Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de la Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, Illkirch, France.
Department of Functional Genomics and Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de la Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, Illkirch, France.
PLoS One. 2017 Jan 26;12(1):e0171043. doi: 10.1371/journal.pone.0171043. eCollection 2017.
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.
视黄酸（RA）通过核视黄酸受体（RARs）在细胞分化和生长停滞中发挥关键作用，RARs是配体依赖性转录因子。虽然RAR激活的主要触发因素是RA的结合，但受体的磷酸化也已成为一种重要的调节信号。已知RARγN端结构域（NTD）的磷酸化在神经元分化中起作用。在这项工作中，我们研究了RARγ配体结合结构域（LBD）的磷酸化，并提供证据表明LBD的磷酸化状态会影响NTD区域的磷酸化。我们解析了LBD的磷酸模拟突变体（RARγS371E）的X射线结构，并用其进行分子动力学模拟，以表征S371E突变对RARγ结构动力学的影响。结合野生型LBD的模拟结果，我们表明LBD盐桥（特别是R387-D340）的构象平衡受S371E突变影响，这可能影响磷酸化NTD的激酶复合物的募集。分子动力学模拟还表明，该盐桥中的保守突变（R387K）影响LBD的动力学，但不会引起大的构象变化。最后，细胞实验表明，在RARγWT以及S371N、S371E和R387K突变体中，视黄酸对RARγNTD的磷酸化有不同的调节作用。这项多学科研究突出了RARγ的LBD和NTD磷酸化之间的变构偶联，并支持了涉及静电相互作用的结构动力学在RARs活性调节中的重要性。
