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.

• Applicant: Dr. Frank Bernhard
• Subject Area: Structural Biology ; Pharmacology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 522414670

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.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Volker Dötsch