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.

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） [Schwarz等人，2007,Bernhard & Tozawa, 2013， Köck等人，2022]。由于它们参与了大量的人类疾病，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见解以了解GPCR功能的必要工具。然而，对洗涤剂的敏感性、固有构象动力学的不稳定性以及特定二硫桥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，我们现在能够生成高质量的全长GPCR和GPCR/ g -蛋白复合物样品，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共翻译插入到含有定制脂质组成膜的预制纳米盘（NDs）中。高亲和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精简的纯化策略相一致，使Cryo-EM样品制备在几个mL的反应体积和不到24小时。除了处理由传统工程稳定的gpcr，如人类β1-肾上腺素能受体（h - β 1ar），新开发的工艺还允许合成迄今为止尚未开发和较少工程的靶标。我们提出了热稳定hβ1AR全长构建体的结构和功能评价，以及野生型人组胺2受体（H2R）和人游离脂肪酸2受体（FFAR2）与g蛋白复合物和脂质环境中的结构和功能评价。通过对所有三种GPCR复合物进行初步冷冻电镜研究，提供了初步的3d密度图，证明了该项目的可行性。结构方法将通过实施新建立的快速纳米转移技术的综合功能研究来支持，该技术可以同时分析体外和培养活细胞中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）脂质环境下的第一个GPCR/Gαsβ1γ2结构，（iii） H2R， FFAR2和hβ1AR的活性构象结构及其同源g蛋白复合物，（IV）制备GPCR二聚体的程序，以及(V) CF合成GPCR转移到细胞环境后功能表征的初步研究。

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.