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- PDB-6whc: CryoEM Structure of the glucagon receptor with a dual-agonist peptide -

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Basic information

Entry
Database: PDB / ID: 6whc
TitleCryoEM Structure of the glucagon receptor with a dual-agonist peptide
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Dual-agonist peptide
  • Glucagon receptor
  • Nanobody35
KeywordsMEMBRANE PROTEIN / GPCR / receptor complex / agonist
Function / homology
Function and homology information


regulation of glycogen metabolic process / glucagon receptor activity / cellular response to glucagon stimulus / exocytosis / response to starvation / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / mu-type opioid receptor binding / developmental growth ...regulation of glycogen metabolic process / glucagon receptor activity / cellular response to glucagon stimulus / exocytosis / response to starvation / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / mu-type opioid receptor binding / developmental growth / corticotropin-releasing hormone receptor 1 binding / intracellular transport / D1 dopamine receptor binding / Hedgehog 'off' state / beta-2 adrenergic receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase activator activity / cellular response to starvation / hormone-mediated signaling pathway / response to nutrient / guanyl-nucleotide exchange factor activity / trans-Golgi network membrane / generation of precursor metabolites and energy / insulin-like growth factor receptor binding / ionotropic glutamate receptor binding / G-protein beta/gamma-subunit complex binding / bone development / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / cognition / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / regulation of blood pressure / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / platelet aggregation / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / adenylate cyclase-activating dopamine receptor signaling pathway / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / G-protein beta-subunit binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / sensory perception of smell / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / glucose homeostasis / retina development in camera-type eye / phospholipase C-activating G protein-coupled receptor signaling pathway / Ca2+ pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / cell population proliferation / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell surface receptor signaling pathway / endosome / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / positive regulation of gene expression / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
GPCR, family 2, glucagon receptor / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / GPCR, family 2, glucagon-like peptide-1/glucagon receptor / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / GPCR family 2, extracellular hormone receptor domain superfamily ...GPCR, family 2, glucagon receptor / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / GPCR, family 2, glucagon-like peptide-1/glucagon receptor / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / G-protein alpha subunit, group S / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / P-loop containing nucleotide triphosphate hydrolases / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Glucagon receptor / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBelousoff, M.J. / Sexton, P. / Danev, R.
Funding support Australia, Japan, China, 10items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1120919 Australia
National Health and Medical Research Council (NHMRC, Australia)1159006 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
Japan Science and Technology#18069571 Japan
Takeda Science Foundation2019 Medical Research Grant Japan
National Science Foundation (NSF, China)2018ZX09735-001 China
National Science Foundation (NSF, China)2018ZX09711002-002-005 China
National Science Foundation (NSF, China)2018ZX09711002-001 China
National Key R&D Program of China2018YFA0507000 China
Novo Nordisk FoundationNNCAS-2017-1-CC China
CitationJournal: J Biol Chem / Year: 2020
Title: Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide.
Authors: Rulue Chang / Xin Zhang / Anna Qiao / Antao Dai / Matthew J Belousoff / Qiuxiang Tan / Lijun Shao / Li Zhong / Guangyao Lin / Yi-Lynn Liang / Limin Ma / Shuo Han / Dehua Yang / Radostin ...Authors: Rulue Chang / Xin Zhang / Anna Qiao / Antao Dai / Matthew J Belousoff / Qiuxiang Tan / Lijun Shao / Li Zhong / Guangyao Lin / Yi-Lynn Liang / Limin Ma / Shuo Han / Dehua Yang / Radostin Danev / Ming-Wei Wang / Denise Wootten / Beili Wu / Patrick M Sexton /
Abstract: Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for ...Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-G complex and compared this structure to our recently published structure of the GCGR-G complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
History
DepositionApr 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 27, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: Dual-agonist peptide
N: Nanobody35
R: Glucagon receptor


Theoretical massNumber of molelcules
Total (without water)163,5576
Polymers163,5576
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15670 Å2
ΔGint-91 kcal/mol
Surface area50190 Å2

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABC

#1: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 45683.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37416.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein/peptide / Antibody / Protein , 3 types, 3 molecules ENR

#4: Protein/peptide Dual-agonist peptide


Mass: 3385.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Antibody Nanobody35


Mass: 15140.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#6: Protein Glucagon receptor / GL-R


Mass: 54068.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GCGR / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P47871

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Glucagon Receptor complexCOMPLEXall0RECOMBINANT
2Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Receptor activity-modifying protein 3, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2, Calcitonin gene-related peptide type 1 receptor, Glucagon ReceptorCOMPLEX#1-#3, #61RECOMBINANT
3Dual-agonist peptideCOMPLEX#41RECOMBINANT
4Nanobody 35COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Lama glama (llama)9844
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
24Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038931
ELECTRON MICROSCOPYf_angle_d0.67712106
ELECTRON MICROSCOPYf_dihedral_angle_d5.9095273
ELECTRON MICROSCOPYf_chiral_restr0.0471348
ELECTRON MICROSCOPYf_plane_restr0.0051555

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