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- EMDB-36324: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-boun... -

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Entry
Database: EMDB / ID: EMD-36324
TitleCryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-bound human GCGR-Gs complex
Map data
Sample
  • Complex: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-bound human GCGR-Gs complex
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Peptide 15
    • Protein or peptide: Nanobody 35Single-domain antibody
    • Protein or peptide: Glucagon receptor
KeywordsG protein-coupled receptor / ligand recognition / receptor activation / unimolecular dual agonist / STRUCTURAL PROTEIN
Function / homology
Function and homology information


regulation of glycogen metabolic process / glucagon receptor activity / G-protein activation / Activation of the phototransduction cascade / : / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 ...regulation of glycogen metabolic process / glucagon receptor activity / G-protein activation / Activation of the phototransduction cascade / : / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Activation of G protein gated Potassium channels / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (z) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Glucagon-type ligand receptors / alkylglycerophosphoethanolamine phosphodiesterase activity / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / G alpha (q) signalling events / photoreceptor outer segment membrane / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / spectrin binding / Vasopressin regulates renal water homeostasis via Aquaporins / response to starvation / cellular response to glucagon stimulus / exocytosis / peptide hormone binding / photoreceptor outer segment / cardiac muscle cell apoptotic process / photoreceptor inner segment / cellular response to starvation / hormone-mediated signaling pathway / guanyl-nucleotide exchange factor activity / response to nutrient / generation of precursor metabolites and energy / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Glucagon signaling in metabolic regulation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Glucagon-type ligand receptors / regulation of blood pressure / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / sensory perception of taste / G-protein beta-subunit binding / heterotrimeric G-protein complex / signaling receptor complex adaptor activity / glucose homeostasis / retina development in camera-type eye / GTPase binding / cell body / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / cellular response to hypoxia / G alpha (s) signalling events / G alpha (q) signalling events / cell population proliferation / cell surface receptor signaling pathway / endosome / G protein-coupled receptor signaling pathway / GTPase activity / dendrite / protein-containing complex binding / positive regulation of gene expression / membrane / plasma membrane / cytoplasm
Similarity search - Function
GPCR, family 2, glucagon receptor / 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, glucagon receptor / 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, 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 / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Glucagon receptor / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human) / Rattus norvegicus (Norway rat) / Bos taurus (cattle) / synthetic construct (others) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsYang L / Zhou QT / Dai AT / Zhao FH / Chang RL / Ying TL / Wu BL / Yang DH / Wang MW / Cong ZT
Funding support China, 8 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81872915 China
National Natural Science Foundation of China (NSFC)82073904 China
National Natural Science Foundation of China (NSFC)82273961 China
National Natural Science Foundation of China (NSFC)32200576 China
National Natural Science Foundation of China (NSFC)82273985 China
National Natural Science Foundation of China (NSFC)82121005 China
National Natural Science Foundation of China (NSFC)81973373 China
National Natural Science Foundation of China (NSFC)21704064 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural analysis of the dual agonism at GLP-1R and GCGR.
Authors: Yang Li / Qingtong Zhou / Antao Dai / Fenghui Zhao / Rulue Chang / Tianlei Ying / Beili Wu / Dehua Yang / Ming-Wei Wang / Zhaotong Cong /
Abstract: Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR), two members of class B1 G protein-coupled receptors, play important roles in glucose homeostasis and energy metabolism. They ...Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR), two members of class B1 G protein-coupled receptors, play important roles in glucose homeostasis and energy metabolism. They share a high degree of sequence homology but have different functionalities. Unimolecular dual agonists of both receptors developed recently displayed better clinical efficacies than that of monotherapy. To study the underlying molecular mechanisms, we determined high-resolution cryo-electron microscopy structures of GLP-1R or GCGR in complex with heterotrimeric G protein and three GLP-1R/GCGR dual agonists including peptide 15, MEDI0382 (cotadutide) and SAR425899 with variable activating profiles at GLP-1R versus GCGR. Compared with related structures reported previously and supported by our published pharmacological data, key residues responsible for ligand recognition and dual agonism were identified. Analyses of peptide conformational features revealed a difference in side chain orientations within the first three residues, indicating that distinct engagements in the deep binding pocket are required to achieve receptor selectivity. The middle region recognizes extracellular loop 1 (ECL1), ECL2, and the top of transmembrane helix 1 (TM1) resulting in specific conformational changes of both ligand and receptor, especially the dual agonists reshaped ECL1 conformation of GLP-1R relative to that of GCGR, suggesting an important role of ECL1 interaction in executing dual agonism. Structural investigation of lipid modification showed a better interaction between lipid moiety of MEDI0382 and TM1-TM2 cleft, in line with its increased potency at GCGR than SAR425899. Together, the results provide insightful information for the design and development of improved therapeutics targeting these two receptors simultaneously.
History
DepositionMay 27, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36324.png

Downloads & links

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Map

FileDownload / File: emd_36324.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0137
Minimum - Maximum-0.03450246 - 0.064458
Average (Standard dev.)0.00016717285 (±0.00168693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36324_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36324_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-boun...

EntireName: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-bound human GCGR-Gs complex
Components
  • Complex: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-bound human GCGR-Gs complex
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Peptide 15
    • Protein or peptide: Nanobody 35Single-domain antibody
    • Protein or peptide: Glucagon receptor

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Supramolecule #1: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-boun...

SupramoleculeName: Cryo-EM structure of the GLP-1R/GCGR dual agonist Peptide 15-bound human GCGR-Gs complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 1
Details: There is no appropriate UniProt/GenBank entry for entity 1 because the protein sequence (P63092) was modified.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.701691 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALTEDEGQ R NEEKAQRE ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALTEDEGQ R NEEKAQRE ANKKIEKQYF LQKIDVIKQA VYRATHRLLL RCRVLGAGIF ETKFQVDKVN FHMFDVGAQR DERRKWIQCF ND VTAIIFV VASSSYNMVI REDNQTNRLQ AALKLFDSIW NNKWLRDTSV ILFLNKQDLL AEKVLAGKSK IEDYFPEFAR YTT PEDATP EPGEDPRVTR AKYFIRDEFL RISTASGDGR HYCYPHFTCA VDTENIRRVF NDCRDIIQRM HLRQYELL

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 37.915496 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSLLQSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD ...String:
MGSLLQSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD TTCALWDIET GQQTTTFTGH TGDVMSLSLA PDTRLFVSGA CDASAKLWDV REGMCRQTFT GHESDINAIC FF PNGNAFA TGSDDATCRL FDLRADQELM TYSHDNIICG ITSVSFSKSG RLLLAGYDDF NCNVWDALKA DRAGVLAGHD NRV SCLGVT DDGMAVATGS WDSFLKIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 7.729947 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
ASNNTASIAQ ARKLVEQLKM EANIDRIKVS KAAADLMAYC EAHAKEDPLL TPVPASENPF REKKFFCAIL

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #4: Peptide 15

MacromoleculeName: Peptide 15 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.385689 KDa
SequenceString:
HSQGTFTSDY SKYLDEQAAK EFIAWLMNT

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Macromolecule #5: Nanobody 35

MacromoleculeName: Nanobody 35 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.343019 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQVQLQESG GGLVQPGGSL RLSCAASGFT FSNYKMNWVR QAPGKGLEWV SDISQSGASI SYTGSVKGRF TISRDNAKNT LYLQMNSLK PEDTAVYYCA RCPAPFTRDC FDVTSTTYAY RGQGTQVTVS SHHHHHHEPE A

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Macromolecule #6: Glucagon receptor

MacromoleculeName: Glucagon receptor / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.627262 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: QVMDFLFEKW KLYGDQCHHN LSLLPPPTEL VCNRTFDKYS CWPDTPANTT ANISCPWYLP WHHKVQHRFV FKRCGPDGQW VRGPRGQPW RDASQCQMDG EEIEVQKEVA KMYSSFQVMY TVGYSLSLGA LLLALAILGG LSKLHCTRNA IHANLFASFV L KASSVLVI ...String:
QVMDFLFEKW KLYGDQCHHN LSLLPPPTEL VCNRTFDKYS CWPDTPANTT ANISCPWYLP WHHKVQHRFV FKRCGPDGQW VRGPRGQPW RDASQCQMDG EEIEVQKEVA KMYSSFQVMY TVGYSLSLGA LLLALAILGG LSKLHCTRNA IHANLFASFV L KASSVLVI DGLLRTRYSQ KIGDDLSVST WLSDGAVAGC RVAAVFMQYG IVANYCWLLV EGLYLHNLLG LATLPERSFF SL YLGIGWG APMLFVVPWA VVKCLFENVQ CWTSNDNMGF WWILRFPVFL AILINFFIFV RIVQLLVAKL RARQMHHTDY KFR LAKSTL TLIPLLGVHE VVFAFVTDEH AQGTLRSAKL FFDLFLSSFQ GLLVAVLYCF LNKEVQSELR RRWHRWRLGK VLWE ERNTS

UniProtKB: Glucagon receptor

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6lmk

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 175000

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