Structural and functional characterization of G-protein-coupled receptors from different classes

G-protein-coupled receptors (GPCRs) are the largest family of surface receptors in the human genome and have been divided into five major families based on their homology and functional characteristics. GPCRs are also called 7-transmembrane receptors and can be activated by various ligands (endogenous or external environmental stimuli), leading to signal transduction through coupling to G proteins. Endogenous ligands bind to the orthosteric binding pocket, while allosteric ligands are ligands that bind any other receptor pocket and modulate the receptor activity allosterically. Receptors are known to be involved in the regulation of various physiological processes in the human body. Currently, more than 30% of FDA-approved drugs are designed to target GPCRs, highlighting their importance to human health. The mechanism of activation, which involves ligand binding and conformational changes that enable coupling with G-protein, varies among different classes of GPCRs. However, our knowledge of how the mechanism of action proceeds and how allosteric modulators affect receptor function is still limited. This work aimed to advance our knowledge of GPCRs, by:  solving the cryo-EM structure of the muscarinic acetylcholine receptor M2 (Class A) with allosteric modulators (intracellular small molecule AW12 and extracellular nanobody NBB4) to better understand the receptor allostery  preparation of a reliable and high-throughput cell-based assay for screening new ligands for the glucagon receptor (Class B) to find a small-molecule agonist for this receptor  preparation of a minimal-cysteine variant and selected double-cysteine mutants of the GABAB receptor (Class C) for future spectroscopic studies to understand the conformational landscape of the receptor The results obtained in this study are divided into three chapters, each devoted to a separate receptor and related problem. Chapter 3 describes the screening of various available strategies to obtain the cryo-EM structure of the muscarinic acetylcholine receptor M2 with allosteric modulators (the small-molecule AW12 developed in computational lab of Prof. Kolb, and the nanobody NBB4 developed in the lab of Prof. Kruse). The final strategy allowed to solve the receptor structure at a resolution of ~4 Å. By stabilizing the receptor with a superagonist and the novel extracellular nanobody NBB4, this work provided important information on allosteric modulation of the receptor, which is consistent with the current literature reporting on M2R allostery. Unfortunately, the small molecule AW12 has not yet been identified. Chapter 4 describes the establishment of a high-throughput BRET-based assay for screening of computationally selected ligands for the glucagon receptor. Although no active binders were identified from the first set of computationally selected compounds, the assay remains crucial for future studies by providing insights into receptor-mediated Go1-protein dissociation. Additionally, the development of a novel Gαo1-Gαs chimeric sensor improved the assay by increasing the BRET amplitude compared to Gs or Go1 alone. This platform provides a promising avenue for testing GCGR targeted ligands to identify new modulators. Chapter 5 focuses on the GABAB receptor. Since little is known about the dynamics of Class C GPCRs dimers, the study obtained a minimal-cysteine (mC) variant of the GABAB, for future spectroscopic studies that may reveal the receptor's conformational landscape. This mC variant dimerizes and expresses at a similar level to the WT. Moreover, functional studies confirm the ability of the mC to bind ligand and couple to G-protein, similarly to the WT receptor, making it a first reported minimalcysteine version of GABAB receptor. This construct is a suitable platform for introducing cysteines at positions of interest for future spectroscopic studies. The established assays that allow for the study of the receptor expression and function also allow for the testing of selected double cys-mutants for spin labeling. Additionally, in this study, it was possible to prepare baculoviruses for future receptor overexpression in Sf9 cells and subsequent purification.