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- PDB-8ykd: Cryo-EM structure of ADGRG2-Gs complex with NTF nanobody -

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Basic information

Entry
Database: PDB / ID: 8ykd
TitleCryo-EM structure of ADGRG2-Gs complex with NTF nanobody
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Adhesion G-protein coupled receptor G2
  • Nanobody-35
  • ScFv-16
KeywordsMEMBRANE PROTEIN / GPCR
Function / homology
Function and homology information


Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / sensory perception of chemical stimulus / Activation of the phototransduction cascade / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / beta-2 adrenergic receptor binding / Activation of G protein gated Potassium channels / G-protein activation ...Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / sensory perception of chemical stimulus / Activation of the phototransduction cascade / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / beta-2 adrenergic receptor binding / 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 / G alpha (12/13) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / G alpha (q) signalling events / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / spermatid development / D1 dopamine receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / insulin-like growth factor receptor binding / ionotropic glutamate receptor binding / adenylate cyclase activator activity / G protein-coupled receptor activity / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / photoreceptor disc membrane / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / sensory perception of taste / signaling receptor complex adaptor activity / retina development in camera-type eye / GTPase binding / phospholipase C-activating G protein-coupled receptor signaling pathway / cell surface receptor signaling pathway / cell population proliferation / apical plasma membrane / G protein-coupled receptor signaling pathway / GTPase activity / synapse / GTP binding / protein-containing complex binding / metal ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
GAIN domain superfamily / GPCR proteolysis site, GPS, motif / : / GPS motif / GAIN-B domain profile. / G-protein-coupled receptor proteolytic site domain / 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) ...GAIN domain superfamily / GPCR proteolysis site, GPS, motif / : / GPS motif / GAIN-B domain profile. / G-protein-coupled receptor proteolytic site domain / 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 / 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 / G protein beta WD-40 repeat protein / 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. / 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
3-BETA-HYDROXY-5-ANDROSTEN-17-ONE / Guanine nucleotide-binding protein subunit gamma / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha / Adhesion G-protein coupled receptor G2
Similarity search - Component
Biological speciesSpodoptera (butterflies/moths)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHuang, S.M. / Sheng-chao, G.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Chem Biol / Year: 2025
Title: Development of an allosteric adhesion GPCR nanobody with therapeutic potential.
Authors: Yuan Zheng / Dan Jiang / Yan Lu / Chao Zhang / Shen-Ming Huang / Haocheng Lin / Daolai Zhang / Shengchao Guo / Jifei Han / Jun Chen / Yaxuan He / Mingxiang Zhang / Yanhui Gao / Yongyuan Guo ...Authors: Yuan Zheng / Dan Jiang / Yan Lu / Chao Zhang / Shen-Ming Huang / Haocheng Lin / Daolai Zhang / Shengchao Guo / Jifei Han / Jun Chen / Yaxuan He / Mingxiang Zhang / Yanhui Gao / Yongyuan Guo / Ran Wei / Ming Xia / Yingying Qin / Zhaoqian Liu / Fan Yang / Shaohua Ge / Fan Yi / Xiao Yu / Hui Lin / Peng Xiao / Jin-Peng Sun / Shiqing Feng /
Abstract: Allosteric modulation of receptor responses to endogenous agonists has therapeutic value, maintaining ligand profiles, reducing side effects and restoring mutant responses. Adhesion G-protein-coupled ...Allosteric modulation of receptor responses to endogenous agonists has therapeutic value, maintaining ligand profiles, reducing side effects and restoring mutant responses. Adhesion G-protein-coupled receptors (aGPCRs), with large N termini, are ideal for allosteric modulator development. We designed a nanobody strategy targeting ADGRG2 N-terminal fragments and got a specific nanobody Nb23-bi, which promoted dehydroepiandrosterone (DHEA)-induced ADGRG2 activation and reversed mutant-induced dysfunctions. By combining structural characterization, crosslinking mass spectrometry, mutational analysis and molecular dynamics simulations, we clarified the allosteric mechanism of how the Nb23-bi modulates conformational changes in the DHEA-binding pocket. Animal studies showed that Nb23-bi promoted the response of DHEA in alleviating testicular inflammation and reversing mutant defects. In summary, we developed an allosteric nanobody of ADGRG2 and gained insights into its functions in reversing disease-associated dysfunctions. Our study may serve as a template for developing allosteric modulators of other aGPCRs for biological and therapeutic purposes.
History
DepositionMar 4, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 13, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 20, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(s) subunit alpha
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein subunit gamma
N: Nanobody-35
R: Adhesion G-protein coupled receptor G2
S: ScFv-16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,1527
Polymers232,8636
Non-polymers2881
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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(s) subunit alpha


Mass: 41879.465 Da / Num. of mol.: 1 / Mutation: G236A,A259D,S262D,L272D,A366S,I372A,V375I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: P63091
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Gene: GNB1 / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: P62871
#3: Protein Guanine nucleotide-binding protein subunit gamma


Mass: 12104.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Gene: mRhiFer1_005810 / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: A0A7J7XNR4

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Antibody , 2 types, 2 molecules NS

#4: Antibody Nanobody-35


Mass: 13885.439 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Production host: Baculovirus expression vector pFastBac1-HM
#6: Antibody ScFv-16


Mass: 26640.643 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Production host: Baculovirus expression vector pFastBac1-HM

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Protein / Non-polymers , 2 types, 2 molecules R

#5: Protein Adhesion G-protein coupled receptor G2 / G-protein coupled receptor 64 / Mouse epididymis-specific protein 6 / Me6


Mass: 98934.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera (butterflies/moths) / Gene: Adgrg2, Gpr64, Me6 / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: Q8CJ12
#7: Chemical ChemComp-AND / 3-BETA-HYDROXY-5-ANDROSTEN-17-ONE


Mass: 288.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H28O2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: Complex of ADGRG2 with nanonody and G protein trimer / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Bos taurus (domestic cattle)9913
21Rattus norvegicus (Norway rat)10116
31Homo sapiens (human)9606
41synthetic construct (others)32630
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241188 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0069769
ELECTRON MICROSCOPYf_angle_d1.19913259
ELECTRON MICROSCOPYf_dihedral_angle_d7.0831337
ELECTRON MICROSCOPYf_chiral_restr0.071500
ELECTRON MICROSCOPYf_plane_restr0.011674

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