Antao Dai

Structure of the full-length glucagon class B G-protein-coupled receptor.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen–deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.

Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators

The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants.

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.

Structure of the glucagon receptor in complex with a glucagon analogue

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression.

GLP-1R (glucagon-like peptide-1 receptor) mediates the ‘incretin effect’ and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs.

Two distinct domains of the glucagon-like peptide-1 receptor control peptide-mediated biased agonism

G protein–coupled receptors (GPCRs) can be differentially activated by ligands to generate multiple and distinct downstream signaling profiles, a phenomenon termed biased agonism. The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR and a key drug target for managing metabolic disorders; however, its peptide agonists display biased signaling that affects their relative efficacies. In this study, we combined mutagenesis experiments and mapping of surface mutations onto recently described GLP-1R structures, which revealed two major domains in the GLP-1/GLP-1R/Gs protein active structure that are differentially important for both receptor quiescence and ligand-specific initiation and propagation of biased agonism. Changes to the conformation of transmembrane helix (TM) 5 and TM 6 and reordering of extracellular loop 2 were essential for the propagation of signaling linked to cAMP formation and intracellular calcium mobilization, whereas ordering and packing of residues in TMs 1 and 7 were critical for extracellular signal–regulated kinase 1/2 (pERK) activity. On the basis of these findings, we propose a model of distinct peptide–receptor interactions that selectively control how these different signaling pathways are engaged. This work provides important structural insight into class B GPCR activation and biased agonism.

Crystal structure of the human 5-HT 1B serotonin receptor bound to an inverse agonist

Abstract5-hydroxytryptamine (5-HT, also known as serotonin) regulates many physiological processes through the 5-HT receptor family. Here we report the crystal structure of 5-HT1B subtype receptor (5-HT1BR) bound to the psychotropic serotonin receptor inverse agonist methiothepin (MT). Crystallization was facilitated by replacing ICL3 with a novel optimized variant of BRIL (OB1) that enhances the formation of intermolecular polar interactions, making OB1 a potential useful tool for structural studies of membrane proteins. Unlike the agonist ergotamine (ERG), MT occupies only the conserved orthosteric binding pocket, explaining the wide spectrum effect of MT on serotonin receptors. Compared with ERG, MT shifts toward TM6 and sterically pushes residues W3276.48, F3306.50 and F3316.51 from inside the orthosteric binding pocket, leading to an outward movement of the extracellular end and a corresponding inward shift of the intracellular end of TM6, a feature shared by other reported inactive G protein-coupled receptor (GPCR) structures. Together with the previous agonist-bound serotonin receptor structures, the inverse agonist-bound 5-HT1BR structure identifies a basis for the ligand-mediated switch of 5-HT1BR activity and provides a structural understanding of the inactivation mechanism of 5-HT1BR and some other class A GPCRs, characterized by ligand-induced outward movement of the extracellular end of TM6 that is coupled with inward movement of the cytoplasmic end of this helix.

High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening

High-throughput identification of GPCR modulators through affinity MS screening.

Design, synthesis, structure–activity relationships, and docking studies of pyrazole-containing derivatives as a novel series of potent glucagon receptor antagonists

Glucagon receptor antagonists possess a great potential for treatment of type 2 diabetes mellitus. A series of pyrazole-containing derivatives were designed, synthesized and evaluated by biological assays as glucagon receptor antagonists. Most of the compounds exhibited good in vitro efficacy. Two of them, compounds 17f and 17k, displayed relatively potent antagonist effects on glucagon receptors with IC50 values of 3.9 and 3.6μM, respectively. The possible binding modes of 17f and 17k with the cognate receptor were explored by molecular docking simulation.