Structural Biology of Multi-Domain Nuclear Receptor Complexes

项目摘要 包括类固醇激素、类维生素A、脂肪酸和膳食脂质在内的亲脂分子控制生殖， 通过直接结合和调节核受体的活性来调节发育和代谢过程 （NRs）。NR与DNA结合并调节基因程序的表达，这些基因程序导致对DNA的生理反应。 它们的小分子配体在过去的几十年里，结构研究主要集中在 配体结合结构域（LBD）或DNA结合结构域（DBD）的NR，但无法揭示如何 多域体系结构被集成在四级结构中。为了了解身体和功能 不同受体结构域的偶联，我们一直在进行涉及全长的X射线衍射研究 和多结构域核受体复合物，其在功能上揭示与DNA，配体， 和辅调节肽。我们以前的研究表明，PPARγ-RXRα的DBDs和LBD 异二聚体和HNF-4α同二聚体通过结构域高度界面排列物理连接 表面，其中一些是DNA依赖的。我们现在建议大大拓宽我们对 核受体家族中的结构域-结构域连接和变构通讯。我们将获得 三个新的NR复合物的晶体结构，包括视黄酸受体（RAR）异二聚体 与维甲酸X受体（RXR）、孕酮受体（PR）同型二聚体和单体Rev-Erbβ 受体的这些不同的NR还辨别由正向重复、反向重复和反向重复组成的不同的反应元件。 重复和单个半位点，因此，与 先前在PPARγ-RXRα或HNF-4α中观察到。我们还建议进行一系列补充性的 生物化学和基于细胞的功能研究，以探测和定量的机制基础， 结构域变构信号在这些受体复合物中的传播。

PROJECT SUMMARY Lipophilic molecules including steroid hormones, retinoids, fatty acids, and dietary lipids control reproductive, developmental and metabolic processes by directly binding and modulating the activities of nuclear receptors (NRs). NRs bind to DNA and regulate the expression of gene programs that lead to physiological responses to their small-molecule ligands. Structural studies over the past few decades have focused primarily on just the ligand binding domains (LBDs) or DNA-binding domains (DBDs) of NRs, but were unable to reveal how the multi-domain architectures are integrated in a quaternary structure. To understand the physical and functional coupling of different receptor domains, we have been conducting X-ray diffraction studies involving full-length and multi-domain nuclear receptor complexes in their functionally revealing complexes bound to DNA, ligands, and coregulator peptides. Our previous studies revealed that DBDs and LBDs of the PPARγ-RXRα heterodimer and HNF-4α homodimer are physically linked through a highly interfaced arrangement of domain surfaces, some of which are DNA-dependent. We now propose to considerably broaden our understanding of the domain-domain connections and allosteric communications in the nuclear receptor family. We will obtain the crystal structures of three new NR complexes that include the Retinoic-Acid Receptor (RAR) heterodimer with Retinoid X Receptor (RXR), the progesterone receptor (PR) homodimer, and the monomeric Rev-Erbβ receptor. These differing NRs also discern distinct response elements consisting of direct repeats, inverted repeats, and single half-sites, thus, distinctive domain-domain interfaces are anticipated as compared to previously seen in PPARγ-RXRα or HNF-4α. We further propose to conduct a series of complementary biochemical and cell-based functional studies to probe and quantitate the mechanistic underpinning of inter- domain allosteric signal propagation in these receptor complexes.