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- PDB-6vcb: Cryo-EM structure of the Glucagon-like peptide-1 receptor in comp... -

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Basic information

Entry
Database: PDB / ID: 6vcb
TitleCryo-EM structure of the Glucagon-like peptide-1 receptor in complex with G protein, GLP-1 peptide and a positive allosteric modulator
Components
  • (Glucagon-like peptide ...) x 2
  • (Guanine nucleotide-binding protein ...) x 3
  • Nanobody 35Single-domain antibody
KeywordsSIGNALING PROTEIN/MEMBRANE PROTEIN / G protein-coupled receptor / membrane protein / family B GPCR / diabetes / allosteric modulator / SIGNALING PROTEIN-MEMBRANE PROTEIN complex
Function / homology
Function and homology information


glucagon receptor binding / glucagon-like peptide 1 receptor activity / glucagon receptor activity / : / hormone secretion / positive regulation of blood pressure / negative regulation of execution phase of apoptosis / post-translational protein targeting to membrane, translocation / feeding behavior / regulation of heart contraction ...glucagon receptor binding / glucagon-like peptide 1 receptor activity / glucagon receptor activity / : / hormone secretion / positive regulation of blood pressure / negative regulation of execution phase of apoptosis / post-translational protein targeting to membrane, translocation / feeding behavior / regulation of heart contraction / response to psychosocial stress / cellular response to glucagon stimulus / positive regulation of calcium ion import / positive regulation of insulin secretion involved in cellular response to glucose stimulus / regulation of insulin secretion / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / Synthesis, secretion, and deacylation of Ghrelin / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of gluconeogenesis / cAMP-mediated signaling / protein kinase A signaling / activation of adenylate cyclase activity / adenylate cyclase activator activity / negative regulation of blood pressure / trans-Golgi network membrane / response to activity / positive regulation of peptidyl-threonine phosphorylation / gluconeogenesis / Olfactory Signaling Pathway / G-protein beta/gamma-subunit complex binding / Activation of the phototransduction cascade / bone development / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / hormone activity / 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 / cognition / platelet aggregation / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / positive regulation of GTPase activity / 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 / 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 / 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 / transmembrane signaling receptor activity / sensory perception of smell / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / glucose homeostasis / retina development in camera-type eye / GTPase binding / positive regulation of peptidyl-serine phosphorylation / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / secretory granule lumen / cell population proliferation / Ras protein signal transduction / Extra-nuclear estrogen signaling / learning or memory
Similarity search - Function
Glucagon / : / GPCR, family 2, glucagon-like peptide-1 receptor / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / GPCR, family 2, glucagon-like peptide-1/glucagon receptor / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain ...Glucagon / : / GPCR, family 2, glucagon-like peptide-1 receptor / Glucagon/GIP/secretin/VIP / Peptide hormone / Glucagon / GIP / secretin / VIP family signature. / Glucagon like hormones / 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-QW7 / Pro-glucagon / 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 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSun, B. / Feng, D. / Bueno, A. / Kobilka, B. / Sloop, K.
CitationJournal: Nat Chem Biol / Year: 2020
Title: Structural insights into probe-dependent positive allosterism of the GLP-1 receptor.
Authors: Ana B Bueno / Bingfa Sun / Francis S Willard / Dan Feng / Joseph D Ho / David B Wainscott / Aaron D Showalter / Michal Vieth / Qi Chen / Cynthia Stutsman / Betty Chau / James Ficorilli / ...Authors: Ana B Bueno / Bingfa Sun / Francis S Willard / Dan Feng / Joseph D Ho / David B Wainscott / Aaron D Showalter / Michal Vieth / Qi Chen / Cynthia Stutsman / Betty Chau / James Ficorilli / Francisco J Agejas / Graham R Cumming / Alma Jiménez / Isabel Rojo / Tong Sun Kobilka / Brian K Kobilka / Kyle W Sloop /
Abstract: Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state ...Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a 'molecular glue'. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric G. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein-protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.
History
DepositionDec 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 30, 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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Assembly

Deposited unit
R: Glucagon-like peptide 1 receptor
P: Glucagon-like peptide 1
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
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody 35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,1477
Polymers162,6676
Non-polymers4801
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16170 Å2
ΔGint-78 kcal/mol
Surface area49280 Å2

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Components

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Glucagon-like peptide ... , 2 types, 2 molecules RP

#1: Protein Glucagon-like peptide 1 receptor / Glucagon-like peptide-1 receptor / GLP-1R


Mass: 51233.109 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P43220
#2: Protein/peptide Glucagon-like peptide 1 / Glucagon-like peptide-1


Mass: 3359.699 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01275

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

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


Mass: 44326.160 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63092
#4: 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#5: 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Antibody / Non-polymers , 2 types, 2 molecules N

#6: 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)
#7: Chemical ChemComp-QW7 / 1-[(1R)-1-(2,6-dichloro-3-methoxyphenyl)ethyl]-6-{2-[(2R)-piperidin-2-yl]phenyl}-1H-benzimidazole


Mass: 480.429 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H27Cl2N3O / 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, GLP-1 peptide and a positive allosteric modulatorCOMPLEX#1-#60RECOMBINANT
3Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, 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-2COMPLEX#3-#51RECOMBINANT
2Glucagon-like peptide 1 receptor, Glucagon-like peptide 1Glucagon-like peptide-1 receptorCOMPLEX#1-#21RECOMBINANT
4Nanobody 35Single-domain antibodyCOMPLEX#61RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Spodoptera frugiperda (fall armyworm)7108
12synthetic construct (others)32630
34Escherichia coli (E. coli)562
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, UltrAuFoil, 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
12RELION33D reconstruction
13PHENIX1.13model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 323427 / 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_d0.00639283
ELECTRON MICROSCOPYf_angle_d0.990112605
ELECTRON MICROSCOPYf_chiral_restr0.05981404
ELECTRON MICROSCOPYf_plane_restr0.00671599
ELECTRON MICROSCOPYf_dihedral_angle_d12.74625457

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