Summary Glucagon-like peptide-1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to GLP-1R, a family B G protein-coupled receptor (GPCR) signaling primarily through the stimulatory G protein Gs. Family B GPCRs are important therapeutic targets, however our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we show the electron cryo-microscopy structure of the peptide-activated GLP-1R:Gs complex at near atomic resolution. The peptide is clasped between the N-terminal domain and transmembrane core of the receptor, further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5 helix of GαsRas. These results provide a structural framework for understanding family B receptor activation through hormone binding.

SUMMARY Class B G protein-coupled receptors are major targets for treatment of chronic diseases, including osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαβγs protein determined by Volta phase plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor through binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G protein coupling via interaction with the Gβ subunit. This structure provides a new framework for understanding G protein-coupled receptor function.

Many important natural products are produced by multidomain nonribosomal peptide synthetases (NRPSs)1–4. During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighboring catalytic domains in an assembly line fashion5. Understanding the structural basis for catalysis with NRPSs will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-NRPSs modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering novel NRPSs.

SUMMARY Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or “megaplexes” more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Metabotropic glutamate receptors are Family C G protein coupled receptors that form obligate dimers and possess extracellular ligand binding Venus flytrap (VFT) domains, which are linked via cysteine rich domains (CRDs) to their 7-transmembrane (7TM) domain. Spectroscopic studies show that signaling is a dynamic process, with large scale conformational changes underlying the transmission of signal from the extracellular VFTs to the G protein-coupling domains (7TMs) in the membrane. Using a combination of x-ray crystallography, cryo-electron microscopy and signaling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the VFTs leads to a compaction of the intersubunit dimer interface, thereby bringing the CRDs into close proximity. Interactions between the CRDs and the second extracellular loops of the receptor enable the rigid body repositioning of the 7TM domains, which come into contact with each other to initiate signaling.