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- PDB-6xox: cryo-EM of human GLP-1R bound to non-peptide agonist LY3502970 -

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Basic information

Entry
Database: PDB / ID: 6xox
Titlecryo-EM of human GLP-1R bound to non-peptide agonist LY3502970
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Alpha subunit of Gs with N-terminus swapped with equivalent residues in Gi,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
  • Glucagon-like peptide 1 receptorGlucagon-like peptide-1 receptor
  • Nanobody 35Single-domain antibody
  • single-chain variable fragment scFv16
KeywordsMEMBRANE PROTEIN / Class B GPCR / glucagon-like peptide-1 receptor / G protein nucleotide exchange factor.
Function / homology
Function and homology information


glucagon-like peptide 1 receptor activity / glucagon receptor activity / sensory perception of chemical stimulus / mu-type opioid receptor binding / hormone secretion / corticotropin-releasing hormone receptor 1 binding / positive regulation of blood pressure / post-translational protein targeting to membrane, translocation / regulation of heart contraction / beta-2 adrenergic receptor binding ...glucagon-like peptide 1 receptor activity / glucagon receptor activity / sensory perception of chemical stimulus / mu-type opioid receptor binding / hormone secretion / corticotropin-releasing hormone receptor 1 binding / positive regulation of blood pressure / post-translational protein targeting to membrane, translocation / regulation of heart contraction / beta-2 adrenergic receptor binding / response to psychosocial stress / PKA activation in glucagon signalling / peptide hormone binding / cAMP-mediated signaling / developmental growth / D1 dopamine receptor binding / Hedgehog 'off' state / ionotropic glutamate receptor binding / insulin-like growth factor receptor binding / activation of adenylate cyclase activity / adenylate cyclase activator activity / negative regulation of blood pressure / Olfactory Signaling Pathway / G-protein beta/gamma-subunit complex binding / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / bone development / 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / 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 / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / 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 / signaling receptor complex adaptor activity / transmembrane signaling receptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / positive regulation of cytosolic calcium ion concentration / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / learning or memory / cell surface receptor signaling pathway / apical plasma membrane / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / GPCR, family 2, glucagon-like peptide-1 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, glucagon-like peptide-1 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 alpha subunit, group S / 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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-V6G / Glucagon-like peptide 1 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 / GNAS complex locus / Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSun, B. / Kobilka, B.K. / Sloop, K.W. / Feng, D. / Kobilka, T.S.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist.
Authors: Takahiro Kawai / Bingfa Sun / Hitoshi Yoshino / Dan Feng / Yoshiyuki Suzuki / Masanori Fukazawa / Shunsuke Nagao / David B Wainscott / Aaron D Showalter / Brian A Droz / Tong Sun Kobilka / ...Authors: Takahiro Kawai / Bingfa Sun / Hitoshi Yoshino / Dan Feng / Yoshiyuki Suzuki / Masanori Fukazawa / Shunsuke Nagao / David B Wainscott / Aaron D Showalter / Brian A Droz / Tong Sun Kobilka / Matthew P Coghlan / Francis S Willard / Yoshiki Kawabe / Brian K Kobilka / Kyle W Sloop /
Abstract: Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved ...Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over β-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.
History
DepositionJul 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Alpha subunit of Gs with N-terminus swapped with equivalent residues in Gi,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
E: single-chain variable fragment scFv16
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody 35
R: Glucagon-like peptide 1 receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,0347
Polymers186,1516
Non-polymers8831
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AR

#1: Protein Alpha subunit of Gs with N-terminus swapped with equivalent residues in Gi,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas / Adenylate cyclase-stimulating G alpha protein / Extra large alphas protein / XLalphas


Mass: 43385.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS, GNAS1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5FWY2, UniProt: Q5JWF2
#6: Protein Glucagon-like peptide 1 receptor / Glucagon-like peptide-1 receptor / GLP-1R


Mass: 51233.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GLP1R / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43220

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

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


Mass: 38534.062 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
#4: 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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Antibody , 2 types, 2 molecules EN

#3: Antibody single-chain variable fragment scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Antibody Nanobody 35 / Single-domain antibody


Mass: 17352.498 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)

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-V6G / 3-[(1S,2S)-1-(5-[(4S)-2,2-dimethyloxan-4-yl]-2-{(4S)-2-(4-fluoro-3,5-dimethylphenyl)-3-[3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl]-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carbonyl}-1H-indol-1-yl)-2-methylcyclopropyl]-1,2,4-oxadiazol-5(4H)-one


Mass: 882.956 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C48H48F2N10O5 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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
1Cryo-EM structure of the Glucagon-like peptide-1 receptor in complex with G protein and a non-peptide agonist LY3502970COMPLEX#1-#60RECOMBINANT
2Glucagon-like peptide 1 receptorGlucagon-like peptide-1 receptorCOMPLEX#61RECOMBINANT
3Trimeric stimulatory G proteinCOMPLEX#1-#2, #41RECOMBINANT
4Nanobody 35Single-domain antibodyCOMPLEX#51RECOMBINANT
5Single-chain variable fragment 16COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.15 MDaNO
15
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Lama glama (llama)9844
45Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Spodoptera frugiperda (fall armyworm)7108
12synthetic construct (others)32630
34Escherichia coli (E. coli)562
45Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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 FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingAverage exposure time: 10.8 sec. / Electron dose: 81.56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 54

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.13_2998refinement
PHENIX1.13_2998refinement
EM software
IDNameVersionCategory
4RELION3CTF correction
7PHENIX1.13model fitting
9PHENIX1.13model refinement
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1598390
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 583053 / Symmetry type: POINT
Atomic model buildingB value: 72.5 / Protocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 5VAI

5vai

RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010910885
ELECTRON MICROSCOPYf_angle_d1.077314796
ELECTRON MICROSCOPYf_chiral_restr0.06441641
ELECTRON MICROSCOPYf_plane_restr0.00711869
ELECTRON MICROSCOPYf_dihedral_angle_d12.60936384

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