Structural Analyses of NR5 Orphan Nuclear Receptors

几年来，我们的实验室一直专注于孤儿核受体在内分泌中的作用 发育和生理学，重点是V亚科(NR5)的成员，包括 类固醇分泌因子1(SF-1)、肝脏受体同系物1(LRH-1)和果蝇FTZ-F1。 一般说来，这些受体不仅参与成人的发育活动，还参与发育活动。 功能，包括类固醇和胆汁酸动态平衡。然而，目前尚不清楚NR5是否会 受体依赖于配体。在与罗伯特·弗莱特里克博士的密切合作中 最近，我们获得了配体结合域的高分辨晶体结构 (LBD)到2.4A分辨率，发现LRH-1在 没有配基。现在，我们已经获得了MSF-1 LBD的晶体结构，分辨率为1.2A 与SHP-1多肽结合。对SF-1和LRH-1结构的比较揭示了总体情况 相似之处，也有一些显著的不同之处。相比之下，观察到的空袋 MLRH-1，MSF-1口袋被磷脂磷脂酰甘油(PG)占据。此外， MSF-1中跨越螺旋H2和H3的区域比这两个区域更加无序和灵活 在mLRH-1中发现。在这里，我们建议将我们对NR5受体的结构分析扩展到我们的 首先，我们将评估螺旋H2和H3的差异如何影响受体功能，并获得 结合SF-1/LRH-1和果蝇FTZ-F1的共调节多肽的晶体结构。 我们的第二个目标将解决磷脂作为NR5的真正配体的潜在相关性。 受体使用生化和生理分析。最后，在我们的第三个目标中，我们将侧重于 定义了这些受体在翻译后修饰后发生的结构变化， 包括相思甲基化和磷酸化。我们认为结构和生化研究 这里提出的建议将极大地补充正在出现的生物学框架，以实现这一重要和 与生物医学相关的核受体亚家族。

For several years our laboratory has focused on the role of orphan nuclear receptors in endocrine development and physiology with an emphasis on members of subfamily V (NR5) that includes steroidocjenic factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and the Drosophila Ftz-F1. Broadly speaking, these receptors participate in developmental events as well as in adult functions, including steroid and bile acid homeostasis. However, it has remained unclear if NR5 receptors are ligand-dependent. In a close collaborative effort with Dr. Robert Fletterick's ab at UCSF, we recently obtained the high-resolution crystal structure of the ligand binding domain (LBD) of LRH-1 to 2.4 A resolution, and find that LRH-1 adopts an active conformation in the absence of ligand. Now, we have obtained the crystal structure of mSF-1 LBD to 1.2 A resolution bound by SHP-1 peptide. Comparison of the SF-1 and LRH-1 structures reveals overall similarities, as well as some striking differences. By contrast to the empty pocket observed for mLRH-1, the mSF-1 pocket is occupied by the phospholipid phosphotidyl glycerol (PG). Moreover, the region in mSF-1 spanning helices H2 and H3 is much more disordered and flexible than that found in mLRH-1. Here, we propose to extend our structural analyses of NR5 receptors In our first aim we will assess how differences in helices H2 and H3 affect receptor function, and obtain the crystal structure of coregulatory peptides bound to SF-1/LRH-1, as well as Drosophila, Ftz-F1. Our second aim will address the potential relevance of phospholipids as bona fide ligands for NR5 receptors using biochemical and physiological assays. Finally, in our third aim, we will focus on defining structural changes that occur following posttranslational modification of these receptors, including sumoylation and phosphorylation. We believe that the structural and biochemical studies proposed here will complement greatly the emerging biological framework for this important and biomedical relevant subfamily of nuclear receptors.