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- PDB-9j1p: Cryo-EM structure of the g1:Ox-bound human GLP-1R-Gs complex -

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Basic information

Entry
Database: PDB / ID: 9j1p
TitleCryo-EM structure of the g1:Ox-bound human GLP-1R-Gs complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Glucagon-like peptide 1 receptor
  • Nanobody-35
  • g1:Ox
KeywordsMEMBRANE PROTEIN / g1:Ox
Function / homology
Function and homology information


adenylate cyclase-activating serotonin receptor signaling pathway / glucagon-like peptide 1 receptor activity / glucagon receptor activity / sensory perception of chemical stimulus / hormone secretion / mu-type opioid receptor binding / positive regulation of blood pressure / corticotropin-releasing hormone receptor 1 binding / G-protein activation / Activation of the phototransduction cascade ...adenylate cyclase-activating serotonin receptor signaling pathway / glucagon-like peptide 1 receptor activity / glucagon receptor activity / sensory perception of chemical stimulus / hormone secretion / mu-type opioid receptor binding / positive regulation of blood pressure / corticotropin-releasing hormone receptor 1 binding / 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 / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / post-translational protein targeting to membrane, translocation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Ca2+ pathway / 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 / beta-2 adrenergic receptor binding / 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 / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / 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 / photoreceptor outer segment membrane / G alpha (q) signalling events / spectrin binding / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / response to psychosocial stress / regulation of heart contraction / Vasopressin regulates renal water homeostasis via Aquaporins / alkylglycerophosphoethanolamine phosphodiesterase activity / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / photoreceptor outer segment / intracellular transport / renal water homeostasis / Hedgehog 'off' state / T cell migration / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / response to prostaglandin E / adenylate cyclase-inhibiting serotonin receptor signaling pathway / adenylate cyclase regulator activity / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / negative regulation of blood pressure / ionotropic glutamate receptor binding / cardiac muscle cell apoptotic process / cellular response to glucagon stimulus / insulin-like growth factor receptor binding / regulation of mitotic spindle organization / cAMP-mediated signaling / photoreceptor inner segment / cellular response to forskolin / adenylate cyclase activator activity / regulation of insulin secretion / trans-Golgi network membrane / positive regulation of cholesterol biosynthetic process / Regulation of insulin secretion / negative regulation of inflammatory response to antigenic stimulus / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / G protein-coupled receptor binding / bone development / G-protein beta/gamma-subunit complex binding
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. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / Hormone receptor domain / GPCR family 2, extracellular hormone receptor domain superfamily ...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. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / Hormone receptor domain / 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-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / 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. / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Glucagon-like peptide 1 receptor / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
Bos taurus (domestic cattle)
Lama glama (llama)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsFan, S. / Li, J. / Zhuang, J. / Zhou, Q. / Mai, Y. / Lin, B. / Wang, M.-W. / Wu, C.
Funding support China, 11items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)91853112 China
National Natural Science Foundation of China (NSFC)22174119 China
National Natural Science Foundation of China (NSFC)82273961 China
National Natural Science Foundation of China (NSFC)82073904 China
National Natural Science Foundation of China (NSFC)81872915 China
National Natural Science Foundation of China (NSFC)21521004 China
Other government20720210001
Other government20720220005IQ
Other governmentIRT_17R66
Other government2021ZD0203400
Other governmentZDKJ2021028
CitationJournal: J Am Chem Soc / Year: 2025
Title: Disulfide-Directed Multicyclic Peptides with N-Terminally Extendable α-Helices for Recognition and Activation of G Protein-Coupled Receptors.
Authors: Shihui Fan / Jie Li / Jie Zhuang / Qingtong Zhou / Yiting Mai / Bingni Lin / Ming-Wei Wang / Chuanliu Wu /
Abstract: Many peptide hormones adopt long α-helical structures upon interacting with their cognate receptors but often exhibit flexible conformations when unbound. Strategies that can stabilize long α- ...Many peptide hormones adopt long α-helical structures upon interacting with their cognate receptors but often exhibit flexible conformations when unbound. Strategies that can stabilize long α-helices without disrupting their binding to receptors are still lacking, which hinders progress in their biological applications and drug development. Here, we present an approach that combines rational design with library screening to create and identify a unique disulfide-directed multicyclic peptide (DDMP) scaffold, which could effectively stabilize N-terminally extendable α-helices while displaying exceptional efficiency in disulfide pairing and oxidative folding. This DDMP scaffold was then utilized for stabilizing the α-helical structure of glucagon-like peptide-1 (GLP-1), resulting in a potent GLP-1 receptor (GLP-1R) agonist with a significantly improved α-helicity and proteolytic stability. By incorporating external α-helices into the DDMP scaffold, we can effectively preserve the native N-terminal α-helical structures while allowing for extensive evolution of the C-terminal disulfide-rich domain for enhancing target binding, as demonstrated by the generation of the DDMP-stabilized GLP-1 (g1:Ox). The cryo-electron microscopy structure of the g1:Ox-GLP-1R in complex with heterotrimeric G reveals the molecular basis for the potent binding between g1:Ox and GLP-1R. Specifically, the DDMP moiety establishes additional interactions with the extracellular domain of GLP-1R, which are absent in the case of GLP-1. Thus, this work offers a novel and effective approach for engineering therapeutic peptides and other peptide α-helices, ensuring that both the N- and C-terminal regions remain essential for target recognition and activation.
History
DepositionAug 5, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Feb 26, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
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
P: g1:Ox
R: Glucagon-like peptide 1 receptor


Theoretical massNumber of molelcules
Total (without water)158,8386
Polymers158,8386
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas


Mass: 41879.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1, GNAS / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P63096, UniProt: P63092, UniProt: Q5JWF2
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Mass: 37915.496 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P54311
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2


Mass: 7729.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212

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

#4: Antibody Nanobody-35


Mass: 15343.019 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)
#5: Protein/peptide g1:Ox


Mass: 5109.745 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: Protein Glucagon-like peptide 1 receptor


Mass: 50860.801 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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Details

Has protein modificationY

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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

ComponentName: Cryo-EM structure of the g1 Ox-bound human GLP-1R-Gs complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Rattus norvegicus (Norway rat)10116
41Bos taurus (domestic cattle)9913
51Lama glama (llama)9844
61Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 453117 / Symmetry type: POINT
RefinementHighest resolution: 2.99 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029606
ELECTRON MICROSCOPYf_angle_d0.51813036
ELECTRON MICROSCOPYf_dihedral_angle_d4.7451311
ELECTRON MICROSCOPYf_chiral_restr0.0391445
ELECTRON MICROSCOPYf_plane_restr0.0041661

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