Structural Genomics of Orphan Nuclear Receptors

描述（由申请方提供）：本研究的长期目标是确定剩余孤儿核受体配体结合结构域（LBD）的晶体结构，并揭示这些结构在其各自信号传导途径中的功能意义。核受体是DNA结合和配体依赖性转录因子，调节基因表达，参与广泛的生理过程。LBD是介导核受体配体信号传导的关键结构域。除了配体结合外，LBD还含有二聚化基序和保守的表面，其介导辅激活子和辅抑制子的配体依赖性或独立性募集以进行转录调控。因此，LBD一直是密集的结构研究和药物发现的焦点。在48种人核激素受体LBD中，超过一半的晶体结构已经确定，只有少数孤儿核受体的LBD结构仍有待解决。这些应用的假设是：1）由核激素受体介导的多种信号传导途径的特异性在很大程度上包含在其LBD的结构组分中，以及2）每个LBD的结构将为理解配体识别、受体二聚化和通过每个受体介导特异性信号传导途径的蛋白质相互作用表面的分子基础提供关键信息。我们的具体目标集中于剩余孤儿核受体LBD的结晶和结构测定，特别是1）CAR LBD，2）COUP-TFI或-TFII LBD，3）TR 2或TR 4 LBD，和4）SHP LBD。在结构确定之后，我们将通过仔细检查和分析结构来确定关键结构元件，我们将与Ming-Jer Tsai（贝勒医学院），Steve Kliewer（德克萨斯大学西南医学中心）和Doug Engel（密歇根大学）合作，进行定点诱变和基于细胞的测定，以验证这些关键结构元件的功能意义。重要性：本申请中产生的结构信息将显著增强我们对这些孤儿核受体如何进化为各自的配体依赖性或非依赖性信号传导途径的分子机制的理解，并且可以作为靶向这些受体的药物发现的合理模板。

DESCRIPTION (provided by applicant): The long-term objective of this study is to determine the crystal structures of the remaining orphan nuclear receptor ligand-binding domains (LBDs) and to reveal the functional implications of these structures in their respective signaling pathways. Nuclear receptors are DNA-binding and ligand-dependent transcriptional factors that modulate gene expression involved in a broad spectrum of physiology. The LBD is the key structural domain that mediates the ligand signaling of nuclear receptors. In addition to ligand binding, the LBD contains dimerization motifs and a conserved surface that mediates ligand-dependent or independent recruitment of coactivators and corepressors for transcriptional regulation. The LBD has thus been the focus of intense structural studies and pharmaceutical discovery. Crystal structures of more than half of the 48 human nuclear hormone receptor LBDs have been determined and there are only a few orphan nuclear receptors for which LBD structure remains to be solved. The hypotheses of these applications are: 1) the specificity of diverse signaling pathways mediated by nuclear hormone receptors is in large part contained with n the structural components of their LBDs, and 2) the structure of each LBD will provide key information for understanding the molecular basis of ligand recognition, receptor dimerization, and protein-interacting surfaces that mediate specific signaling pathways by each receptor. Our specific aims are focused on crystallization and structural determination of the remaining orphan nuclear receptor LBDs, particularly, 1) the CAR LBD, 2) the COUP-TFI or -TFII LBD, 3) the TR2 or TR4 LBD, and 4) the SHP LBD. Following the structural determination, we will identify key structural elements by scrutinizing and analyzing the structures, and we will collaborate with Ming-Jer Tsai (Baylor College of Medicine), Steve Kliewer (University of Texas Southwestern Medical Center), and Doug Engel (University of Michigan), on site-directed mutagenesis and cell-based assays to validate the functional significance of these key structural elements. Significance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of how these orphan nuclear receptors have evolved for their respective ligand-dependent or -independent signaling pathways, and can serve as rational templates for drug discovery that targets these receptors.