Signal Transduction Mechanism Study Of Glucagon Receptor

G-protein-coupled receptors(GPCRs)are the largest membrane protein family and represent interesting drug targets.The receptors are activated by peptide and non-peptide neurotransmitter,hormone,growth factor and odorant,and form complex with G protein or arrestins to transmit signaling,thereby influencing human physiological activities.Class B GPCRs are activated by natural peptidic hormone,and play important roles in many chronic disease,such as diabetes,metabolic disease,osteoporosis and nervous system disease.The structure of these receptors is characterized as a large extracellular N-terminal domain(ECD)adjacent to a transmembrane domain(TMD).Glucagon receptor(GCGR)is a member of class B GPCR and activated by glucagon to modulate blood glucose balance,and is an attractive drug target for type 2 diabetes.The TMD structure of GCGR has been determined in 2013.However,due to conformational flexibility and diversity,the determination of full-length structures of class B receptors remain extremely challenging.While ECD and TMD of class B GPCRs both contribute to peptide binding,the knowledge of full-length class B GPCR structures is of great significance for studying peptide binding mode and receptor activation mechanism.In this study,the complex structure of the full-length GCGR in complex with an allosteric inhibitor NNC0640 and an antibody fragment mAb1 was determined at 3?,using X-ray free electron laser(XFEL).The three-dimensional structure of the full-length class B GPCR was determined at atomic level for the first time.The conformational changes at different functional states revealed the regulation mechanism of different domains on receptor activity,expanding the understanding of ligand recognition and activation mechanism of class B GPCR.Our structure also provides high-precision structural template for drug design and development of diabetes mellitus.Wild type full-length GCGR was poor expressed and highly unstable.To improve the protein yield and stability,fusion partners and junction sites,C-terminal truncations and mutants were screened.A negative allosteric modulator NNC0640 and the antigen-binding fragment(Fab)of an inhibitory antibody,mAb1,were added to stabilize the receptor during purification.Lipidic cubic phase(LCP)technique was used for crystallization.Different conditions(precipitant,pH,salt and additive)were screened and the GCGR-NNC0640-mAb1 structure was determined to 3.2?by synchrotron radiation.X-ray free-electron lase was used to improve the resolution up to 3.0?.In the inactive state GCGR-NNC0640-mAb1 structure,small molecular NNC0640 binds to the external surface of TMD,and antibody fragment mAb1stabilizes the ECD.The 12-residue segment termed stalk connecting the ECD and TMD adopts a?-strand conformation,which forms an?-helix in the previously determined structure of GCGR TMD.The first extracellular loop(ECL1),which exhibits a?-hairpin conformation,forms multiple interactions with stalk.The hydrogen-deuterium exchange.Disulfide crosslinking and molecular dynamics illustrate that the stalk and ECL1 play an important role in peptide binding and receptor activation.The complex structure of GCGR and downstream effector protein G_s was then studied to obtain the activated GCGR structure and facilitate understanding of interaction and activation mechanism of agonist peptide with the receptor.Different peptide agonists and experimental protocols have been screened and the data was collected by Tecnai Spirit 120 kV,Talos F200C 200 kV,Titan Krios 300 kV.Finally,cryo-electronic microscopy(cryo-EM)structures of GCGR-G_s-peptide A and GCGR-G_s-peptide D were determined at 4.3?and 3.7?resolution,respectively.In the GCGR-G_s complex structure,the peptides located between ECD and TMD with their N-and C-terminus interacting with TMD and ECD.The?and?subunit of G_s form extensive interactions with receptor to stabilize the active receptor conformation.In summary,the structures of inactivated and activated full-length GCGR determined using X-ray,XFEL and cryo-EM,in combination with functional assays elucidate the signal recognition and receptor activation mechanism of GCGR,which greatly expand our understanding of signal transduction mechanism of class B GPCR and provide an important structural basis for the development of anti-diabetic drugs.