G protein-coupled receptors (GPCRs) are the largest family of signaling proteins in animals and represent the largest family of druggable targets in the human genome. Therefore, it is of no surprise that the molecular mechanisms of GPCR activation and signal transduction have attracted close attention for the past few decades. Several stabilizing interactions within the GPCR transmembrane (TM) domain helices regulate receptor activation. An example is a salt bridge between 2 highly conserved amino acids at the bottom of TM3 and TM6 that has been characterized for a large number of GPCRs. Through structural modeling and molecular dynamics (MD) simulations, we predicted several electrostatic interactions to be involved in metabotropic glutamate receptor 2 (mGlu2R) activation. To experimentally test these predictions, we employed a charge reversal mutagenesis approach to disrupt predicted receptor electrostatic intramolecular interactions as well as intermolecular interactions between the receptor and G proteins. Using two electrode voltage clamp in Xenopus laevis oocytes expressing mutant receptors and G-proteins, we revealed novel electrostatic interactions, mostly located around intracellular loops 2 and 3 of mGlu2R, that are critical for both receptor and G-protein activation. These studies contribute to elucidating the molecular determinants of mGluRs activation and conformational coupling to G-proteins, and can likely be extended to include other classes of GPCRs.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

Rhodopsin ist als Sehpigment der Photorezeptorzellen einer der am aktivsten untersuchten GPCRs. Es besteht aus dem Apoprotein Opsin und dem inversen Agonisten 11-cis-Retinal. Der inaktivierende Ligand ist in der sieben Transmembran- Helix (TM)-Struktur des Rezeptors kovalent gebunden und muss durch Licht cis/trans-isomerisiert werden, um den Rezeptor zu aktivieren. Der aktivierte Rezep-tor katalysiert den Nukleotidaustausch im G-Protein und zerfällt innerhalb von Minuten in Opsin und all-trans-Retinal. Das visuelle Pigment wird dann durch erneute Beladung des Opsins mit 11-cis-Retinal wieder hergestellt. In der vorliegenden Arbeit wird die erfolgreiche Kristallisation des nativen Opsins aus der Stäbchenzelle der Rinderretina und die Bestimmung der Proteinstruktur bei 2.9 Å Auf-lösung dargestellt. Im Vergleich zur bekannten Struktur des inaktiven Rhodopsins zeigt Opsin deutliche Strukturänderungen in den konservierten E(D)RY und NPxxY(x)5,6F Regionen und in TM5-TM7. Auf der intrazellulären Seite ist TM6 ca. 6-7 Å nach außen gekippt, während die TM5 Helix verlängert und näher zu TM6 verschoben ist. Durch die strukturellen Änderungen, von denen einige einem aktiven GPCR Zustand zugeschrieben werden können, wird die leere Retinalbindungstasche reorganisiert, um zwei Öffnungen für Aus- und Eintritt von Retinal bereitzustellen. Die Struktur von Opsin liefert neue Erkenntnisse zur Bindung von hydrophoben Liganden an GPCRs, zur GPCR-Aktivierung und zur Signalübertragung auf das G-Protein. / Rhodopsin as the visual pigment in photoreceptor cells is one of the most actively studied GPCRs. It consists of the apoprotein opsin and the inverse agonist, 11-cis-retinal. The inactivating ligand is bound in the seven-transmembrane helix (TM) bundle and cis/trans-isomerized by light to activate the receptor. The active receptor state is capable of catalyzing nucleotide exchange in the G protein and decays within minutes into opsin and all-trans-retinal. The visual pigment is then restored by reloading opsin with new 11-cis-retinal. In the present work, the successful crystallization of native opsin from bovine retinal rod cells and determination of the protein structure to 2.9 Å resolution is presented. Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x)5,6F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.