Cryo-electron microscopy structures and functional analysis of cell-free synthesized G-protein coupled receptors in complex with their cognate G-proteins and cotranslationally inserted into nanodiscs.

无细胞合成的 G 蛋白偶联受体与其同源 G 蛋白复合并共翻译插入纳米盘的冷冻电子显微镜结构和功能分析。

• 批准号: 522414670
• 负责人: Dr. Frank Bernhard
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/522414670?language=en
• 关键词: Cryo; electron; microscopy; structures; functional

Our group has continuously optimized the cell-free (CF) production of membrane proteins, with a particular focus on G-protein coupled receptors (GPCRs) [Schwarz et al., 2007, Bernhard & Tozawa, 2013, Köck et al., 2022]. Due to their involvement in a plethora of human diseases, the family of GPCRs is of prime interest to the pharmaceutical industry. Efficient production pipelines are therefore essential tools to reveal the necessary structural insights in order to understand GPCR function. However, sensitivity to detergents, instability due to intrinsic conformational dynamics and the essential requirement for specific disulfide bridge formation poses significant problems to sample preparation. By systematically adjusting the reaction conditions and expression strategies in our developed CF expression platform, we are now able to generate high quality samples of full-length GPCRs and GPCR/G-protein complexes in sufficient amounts not only for functional characterization but also for Cryo-electron microscopy (Cryo-EM) studies. Our strategy is the cotranslational insertion of GPCRs into preformed nanodiscs (NDs) containing membranes of tailored lipid composition. The simultaneous presence of high affinity ligands instantly supports and stabilizes GPCR folding and our efficient two-compartment CF system in concert with optimized redox conditions and streamlined purification strategies allows Cryo-EM sample preparation in few mL reaction volumes and in less than 24 hours. Besides addressing GPCRs stabilized by conventional engineering such as the human β1-adrenergic receptor (hβ1AR), the newly developed process allows in addition the synthesis of so far underexplored and less engineered targets. We propose the structural and functional evaluation of full-length constructs of the thermostabilized hβ1AR as well as of wild type versions of the human histamine 2 receptor (H2R) and of the human free fatty acid 2 receptor (FFAR2) in complex with G-proteins and in lipid environment. The feasibility of the project is demonstrated by providing preliminary 3D-density maps already obtained by initial Cryo-EM studies of all three GPCR complexes. The structural approaches will be supported by comprehensive functional studies implementing a newly established fast nanotransfer technique that allows to simultaneously analyze CF synthesized GPCR samples in vitro and in cultured living cells. Expected highlights of the project will be (i) the first high resolution structure of CF synthesized GPCRs, (ii) the first GPCR/Gαsβ1γ2 structures in lipid environment, (iii) the first structures of H2R, FFAR2 and hβ1AR in active conformation and in complex with their cognate G-proteins, (IV) procedures for the preparation of GPCR dimers and (V) pilot studies for the functional characterization of CF synthesized GPCRs after transfer into cellular context.

我们的团队不断优化膜蛋白的无细胞（CF）生产，特别关注G蛋白偶联受体（GPCR）[施瓦茨et al.，2007，Bernhard & Tozawa，2013，Köck et al.，2022年]。由于它们参与过多的人类疾病，GPCR家族是制药行业的主要兴趣。因此，高效的生产管道是揭示必要的结构洞察力以了解气相化学还原功能的重要工具。然而，对洗涤剂的敏感性、由于内在构象动力学的不稳定性以及对特定二硫键形成的基本要求给样品制备带来了重大问题。通过在我们开发的CF表达平台中系统地调整反应条件和表达策略，我们现在能够产生足够量的全长GPCR和GPCR/G蛋白复合物的高质量样品，不仅用于功能表征，而且用于冷冻电子显微镜（Cryo-EM）研究。我们的策略是将GPCR共翻译插入预先形成的含有定制脂质组成的膜的纳米盘（ND）中。高亲和力配体的同时存在立即支持和稳定GPCR折叠，我们高效的双室CF系统与优化的氧化还原条件和简化的纯化策略相结合，允许在几mL反应体积和不到24小时内制备Cryo-EM样品。 除了解决通过常规工程如人β1-肾上腺素能受体（hβ1AR）稳定的GPCR外，新开发的工艺还允许合成迄今为止未开发和较少工程化的靶标。我们提出了热稳定hβ1AR全长构建体以及与G蛋白复合的人组胺2受体（H2 R）和人游离脂肪酸2受体（FFAR 2）的野生型版本和脂质环境中的结构和功能评价。该项目的可行性证明提供初步的三维密度图已经获得了初步的冷冻电镜研究的所有三个GPCR复合物。结构方法将得到全面的功能研究的支持，该研究实施了一种新建立的快速纳米转移技术，该技术允许在体外和培养的活细胞中同时分析CF合成的GPCR样品。该项目的预期亮点将是（i）CF合成GPCR的第一个高分辨率结构，（ii）脂质环境中的第一个GPCR/Gαsβ1γ2结构，（iii）H2 R，FFAR 2和hβ 1 AR的第一个结构，其活性构象及其同源G蛋白的复合物，（IV）制备GPCR二聚体的程序和（V）转移到细胞环境中后CF合成的GPCR的功能表征的初步研究。