Structural Genomics of Orphan Nuclear Receptors

描述(申请人提供)：这项研究的长期目标是确定剩余的孤儿核受体配体结合域(LBD)的晶体结构，并揭示这些结构在各自的信号通路中的功能意义。核受体是DNA结合和配体依赖的转录因子，调节基因表达，涉及广泛的生理过程。LBD是介导核受体配体信号转导的关键结构域。除了配体结合，LBD还含有二聚化基序和一个保守的表面，该表面介导依赖于或独立招募辅助激活因子和辅助抑制因子进行转录调控。因此，LBD一直是紧张的结构研究和药物发现的焦点。在48个人类核激素受体LBD中，一半以上的晶体结构已经确定，只有少数孤儿核受体的LBD结构有待解决。这些应用的假设是：1)核激素受体介导的不同信号通路的特异性在很大程度上取决于它们LBD的结构成分，2)每个LBD的结构将为理解配体识别、受体二聚化和蛋白质相互作用表面的分子基础提供关键信息，这些表面介导每个受体介导特定的信号通路。我们的具体目标集中在剩余的孤儿核受体LBD的结晶和结构测定上，特别是1)CAR LBD，2)COUP-TFI或-TFII LBD，3)TR2或TR4 LBD，以及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的结构元件，我们将与贝勒医学院的Ming-Jer Tsai、德克萨斯大学西南医学中心的Steve Kliewer和密歇根大学的Doug Engel合作，进行定点突变和基于细胞的分析，以验证这些关键结构元件的功能意义。意义：在这一应用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为各自的配体依赖或独立的信号通路进化的分子机制的理解，并可以作为针对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.