Benko Szilvia, Love James D, Beládi Marta, Schwabe John W R, Nagy Laszlo
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Nagyerdei krt. 98, Debrecen H-4012, Hungary.
J Biol Chem. 2003 Oct 31;278(44):43797-806. doi: 10.1074/jbc.M306199200. Epub 2003 Aug 12.
The repressive and activating states of nuclear hormone receptors are achieved through the recruitment of cofactor proteins. The binding of co-repressors and co-activators is believed to be mutually exclusive and principally regulated by ligand binding. To understand the molecular determinants of the switch induced by ligand in the retinoic acid receptor and in particular the intrinsic role of the ligand binding domain (LBD) in cofactor binding and release, we carried out extensive mutational analysis of surface residues of the LBD. As seen previously we found that co-repressor and co-activator molecules bind to overlapping docking sites on the surface of the retinoic acid receptor alpha LBD. Perturbation of this surface impaired both co-activator and co-repressor association resulting in a transcriptionally inert receptor. Unexpectedly mutation of two residues, Trp-225 and Ala-392, which lie outside the docking site, had opposite effects on co-activator and co-repressor binding. W225A was a constitutive repressor that failed to bind co-activator and exhibited an increased, and ligand-insensitive, interaction with co-repressor. A392R, on the other hand, had reduced affinity for co-repressors and increased affinity for co-activators and behaved as a constitutive, but still ligand-inducible, activator. Analysis of known structures showed that these mutations lie in the proximity of helix 12 (H12), and their effects are likely to be the result of perturbations in the behavior of H12. These data suggest that residues in the close vicinity of H12 regulate cofactor affinity and determine the basal activity of receptors.
核激素受体的抑制状态和激活状态是通过募集辅因子蛋白来实现的。共抑制因子和共激活因子的结合被认为是相互排斥的，并且主要受配体结合的调节。为了了解视黄酸受体中配体诱导的开关的分子决定因素，特别是配体结合域（LBD）在辅因子结合和释放中的内在作用，我们对LBD的表面残基进行了广泛的突变分析。如先前所见，我们发现共抑制因子和共激活因子分子结合到视黄酸受体α LBD表面的重叠对接位点上。该表面的扰动损害了共激活因子和共抑制因子的结合，导致转录惰性受体。出乎意料的是，位于对接位点之外的两个残基Trp-225和Ala-392的突变对共激活因子和共抑制因子的结合产生了相反的影响。W225A是一种组成型抑制剂，它不能结合共激活因子，并且与共抑制因子的相互作用增加且对配体不敏感。另一方面，A392R对共抑制因子的亲和力降低，对共激活因子的亲和力增加，表现为组成型但仍可被配体诱导的激活剂。对已知结构的分析表明，这些突变位于螺旋12（H12）附近，它们的影响可能是H12行为扰动的结果。这些数据表明，H12附近的残基调节辅因子亲和力并决定受体的基础活性。
