Harris Jonathan M, Lau Patrick, Chen Shen Liang, Muscat George E O
Queensland University of Technology, Centre for Molecular Biotechnology, Brisbane 4001, Queensland, Australia.
Mol Endocrinol. 2002 May;16(5):998-1012. doi: 10.1210/mend.16.5.0837.
The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of RORalpha using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORalpha ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that RORalpha is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between RORalpha and TRbeta in the way that the AF2 helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORalpha (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest 1) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORalpha Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of RORalpha lacking AF2, and Phe491Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.
类视黄醇孤儿相关受体α（RORα）是孤儿受体ROR亚家族的成员，在没有外源性配体的情况下作为转录的组成型激活剂。为了理解这种活性的基础，我们以密切相关的TRβ为模板构建了RORα的同源模型。分子建模表明，庞大的疏水侧链占据了RORα配体腔，留下一个小而独特的腔，可能参与受体稳定。该模型与来自类固醇受体共激活因子、GR相互作用蛋白-1的受体相互作用肽进行对接模拟，描绘了一个由跨越螺旋3-5和螺旋12的特征基序组成的共激活因子结合表面[激活功能2（AF2）]。用扫描丙氨酸诱变探测该表面显示，RORα和TRβ的同源残基之间存在结构和功能等效性。这很令人惊讶（鉴于RORα是一种不依赖配体的激活剂，而TRβ对配体有绝对需求），并促使我们使用分子建模来确定RORα和TRβ在AF2螺旋与受体其余部分相互作用方式上的差异。建模突出了RORα螺旋11中的一个非保守氨基酸（Phe491）以及AF2 N端的一段短的3.10螺旋，我们认为1）这确保了AF2永久锁定在为其他配体结合受体描述的全构象中，因此2）能够不依赖配体招募共激活因子。与此一致的是，将RORα的Phe491突变为甲硫氨酸或丙氨酸（甲硫氨酸是TRβ中的同源残基），分别降低和消除了转录激活和共激活因子的招募。此外，我们能够通过过表达GAL-AF2融合蛋白来重建缺乏AF2的RORα缺失突变体和Phe491Met的转录活性，证明了AF2不依赖配体的招募以及Phe491在招募AF2中的作用。
