Towards a crystal structure of the Family B GPCR PTH2

B族GPCR PTH2的晶体结构

• 批准号: 1774805
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1774805
• 关键词: Towards; crystal; structure; Family; GPCR

Background: Family B G protein-coupled receptors (GPCRs) are validated drug targets, some already exploited through the development of peptidic drugs (e.g. extenatide at the GLP-1 receptor for type 2 diabetes; teriparatide at PTH1 for osteoporosis). However, the discovery of small molecule ligands for Family GPCRs, particularly agonists, has proven to be intractable. One reason for the lack of success has been the paucity in our understanding of the structure of Family B GPCRs and, in particular, how peptide ligands bind and activate them.Objectives: (1) To overexpress, purify and crystallise PTH2 using the insect cell/baculovirus system (Saarenpää et al., 2015). (2) To use molecular modelling, site-directed mutagenesis and pharmacological assays: (i) To identify key residues in the 7TM domain that contribute to peptide agonist recognition; (ii) To generate a disulphide-trapped conformational state of PTH2 by cross-linking the peptide ligand to both the NTD and 7TM domains of the full-length receptor in order to stabilise the ligand-bound active state. Novelty: There are no known structures for any full-length Family B GPCRs, probably due to the extensive conformational flexibility between the N-terminal domain and the 7TM domain - trapping this state using the ligand is a novel approach. The PTH2 receptor is unexplored from a structure-function perspective.Timeliness: GPCR structure determination is in a golden period but Family B GPCRs are devoid of known structure - progress in this area is important.Experimental Approach: (1) The insect cell/baculovirus system is already in use in the Goldman laboratory. Virus expressing the PTH2 cDNA will be generated and used for over-expression and purification of PTH2 protein. (2) A molecular model of the full-length peptide-bound PTH2 has already been generated by Donnelly for the planning of site-directed mutagenesis targets. All the necessary pharmacological radio-ligand binding and 384-well cAMP assays are established in Donnelly's lab.

背景资料：家族B G蛋白偶联受体（GPCR）是经过验证的药物靶点，其中一些已经通过肽类药物的开发而得到利用（例如，针对GLP-1受体的extenatide用于治疗2型糖尿病;针对PTH 1的特立帕肽用于治疗骨质疏松症）。然而，家族GPCR的小分子配体，特别是激动剂的发现已被证明是棘手的。缺乏成功的一个原因是我们对家族B GPCR的结构，特别是肽配体如何结合和激活它们的理解不足。2015年）。(2)使用分子建模、定点诱变和药理学测定：（i）鉴定有助于肽激动剂识别的7 TM结构域中的关键残基;（ii）通过将肽配体与全长受体的NTD和7 TM结构域交联以稳定配体结合的活性状态，产生二硫键捕获的PTH 2构象状态。新奇：对于任何全长家族B GPCR，没有已知的结构，可能是由于N-末端结构域和7 TM结构域之间广泛的构象灵活性-使用配体捕获这种状态是一种新的方法。PTH 2受体的结构-功能研究尚处于起步阶段。时间：GPCR结构鉴定正处于黄金时期，但家族B GPCR尚缺乏已知的结构--这方面的进展很重要。实验方法：（1）昆虫细胞/杆状病毒系统已在Goldman实验室使用。将产生表达PTH 2 cDNA的病毒并用于PTH 2蛋白的过表达和纯化。(2)全长肽结合的PTH 2的分子模型已经由Donnelly产生，用于规划定点诱变靶点。所有必要的药理学放射性配体结合和384孔cAMP测定都在Donnelly的实验室中建立。