Full-length LRH-1 structural regulation

全长LRH-1结构调整

• 批准号: 10034145
• 负责人: Raymond Daniel Blind
• 金额: $ 34.6万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10034145
• 关键词: Address; Affect; Affinity; Agonist; Amino Acids; Architecture; Benzophenones; Bile Acids; Binding; Biophysics; Chemicals; Cholesterol; Cholesterol Homeostasis; Clinic; Clinical; Communication; Computer Models; Cryoelectron Microscopy; Crystallography; DNA; DNA Binding Domain; Data; Development; Drug Design; Drug Targeting; Event; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Transcription; Glucose; Hepatocyte; Human; Hydrophobicity; Individual; Knockout Mice; Length; Ligand Binding; Ligand Binding Domain; Ligands; Liver; Metabolic; Modeling; Molecular; Mus; Mutation; NR5A2 gene; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Receptors; Oranges; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Positioning Attribute; Post-Translational Protein Processing; Regulation; Resolution; Structural Models; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Treatment Efficacy; base; biophysical analysis; biophysical techniques; blood glucose regulation; cohesion; crosslink; cysteinylcysteine; drug development; experience; flexibility; insight; liver metabolism; mouse model; mutant; nanomolar; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; optimism; pre-clinical; prevent; receptor function; recruit; response; simulation; small molecule; structural biology; tool

LRH-1 (NR5A2) is a monomeric nuclear receptor involved in many aspects of liver physiology, including bile acid, cholesterol and glucose homeostasis. LRH-1 activation has beneficial effects on liver metabolism in pre-clinical mouse models. As nuclear receptors like LRH-1 have a very druggable ligand-binding pocket, LRH-1 has been targeted by many drug development efforts with great recent progress, however an LRH-1 agonist is still not available in the clinic. Like most other nuclear receptors, LRH-1 is composed of a DNA-binding domain and a ligand-binding domain, which are connected by a large unstructured Hinge domain. Classic nuclear receptor drug design has focused on the isolated ligand-binding domain, as the regulatory mechanism of this isolated domain is very well understood at the molecular level: binding of a hydrophobic small molecule allosterically alters ligand-binding domain recruitment of a transcriptional coregulator, which regulates nuclear receptor function. However, several lines of evidence suggest inter-domain communication exists between LRH-1 domains, regulating function. Understanding the structural biology behind this inter-domain communication might help LRH-1 drug design efforts, however technical challenges in applying crystallography or cryo-EM has prevented progress, despite great effort from several groups. We used an integrated structural approach to develop a low-resolution, but high confidence model of the intact, full-length LRH-1, using exclusively solution-based biophysical analyses and computational modeling (HDX, SAXS, chemical crosslinking, artificial amino acid benzophenone cross linking, Cys-Cys interdomain crosslinking, Rosetta and MD simulations). The model explains human patient mutations and structure-based mutations predicted to reside in the interface between the domains, which we show alter full length LRH-1 structure and function. Here, we propose to take advantage of this solution-based approach to address several long-standing questions in the field: Aim 1 determines how various ligands change full length LRH-1 interdomain communication. Aim 2 resolves how the SUMO module post-translational modification alters LRH-1 interdomain communication. Aim 3 identifies the genes effected by structure-based LRH-1 mutations in mouse liver and primary hepatocytes. Our current understanding of how LRH-1 structure is regulated is limited to studies of the individual domains. Without understanding how full-length LRH-1 is regulated, we cannot know if our current drug design efforts are taking full advantage of the entire therapeutic capacity of LRH-1.

LRH-1 （NR5A2）是一种单体核受体，参与肝脏生理的许多方面，包括胆汁酸、胆固醇和葡萄糖的稳态。在临床前小鼠模型中，LRH-1激活对肝脏代谢有有益的影响。由于像LRH-1这样的核受体具有非常可药物化的配体结合口袋，LRH-1已成为许多药物开发工作的靶点，近年来取得了很大进展，但临床仍然没有一种LRH-1激动剂。与大多数其他核受体一样，LRH-1由dna结合域和配体结合域组成，它们通过一个大的非结构化Hinge结构域连接。经典的核受体药物设计主要集中在分离的配体结合区域，因为这种分离区域的调控机制在分子水平上已经得到了很好的理解：疏水小分子的结合变构地改变了转录共调节剂的配体结合区域募集，从而调节核受体的功能。然而，一些证据表明LRH-1结构域之间存在域间通信，调节功能。了解这种域间通信背后的结构生物学可能有助于LRH-1药物设计工作，然而，尽管几个小组付出了巨大的努力，但应用晶体学或冷冻电镜技术方面的技术挑战阻碍了进展。研究人员利用基于溶液的生物物理分析和计算建模（HDX、SAXS、化学交联、人工氨基酸二苯甲酮交联、Cys-Cys结构域间交联、Rosetta和MD模拟），采用集成结构方法建立了完整全长LRH-1的低分辨率、高置信度模型。该模型解释了人类患者突变和基于结构的突变预测驻留在域之间的界面，我们显示改变全长LRH-1结构和功能。在这里，我们建议利用这种基于解决方案的方法来解决该领域的几个长期存在的问题：目标1确定各种配体如何改变全长LRH-1域间通信。目标2解决了SUMO模块翻译后修饰如何改变LRH-1域间通信。目的3鉴定小鼠肝脏和原代肝细胞中受LRH-1结构突变影响的基因。我们目前对LRH-1结构如何调控的理解仅限于对单个结构域的研究。如果不了解全长LRH-1是如何被调控的，我们就无法知道我们目前的药物设计工作是否充分利用了LRH-1的整个治疗能力。