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- PDB-9hb3: cryo-EM structure of inactive human arginine-vasopressin (AVP) V2... -

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Basic information

Entry
Database: PDB / ID: 9hb3
Titlecryo-EM structure of inactive human arginine-vasopressin (AVP) V2 receptor (V2R) with Mambaquaretin1 K39A (MQK39A)
Components
  • Mambaquaretin-1
  • Synthetic antibody, anti-BRIL Fab fragment, Heavy chain
  • Synthetic antibody, anti-BRIL Fab fragment, Light chain
  • anti-BRIL Fab Nanobody
  • arginine-vasopressin (AVP) V2 receptor (V2R)-BRIL,Vasopressin V2 receptor,Vasopressin V2 receptor
KeywordsMEMBRANE PROTEIN / GPCR / V2R / TVP / tolvaptan
Function / homology
Function and homology information


renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / hemostasis / telencephalon development / positive regulation of systemic arterial blood pressure / positive regulation of intracellular signal transduction / endocytic vesicle ...renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / hemostasis / telencephalon development / positive regulation of systemic arterial blood pressure / positive regulation of intracellular signal transduction / endocytic vesicle / cellular response to hormone stimulus / activation of adenylate cyclase activity / positive regulation of vasoconstriction / response to cytokine / clathrin-coated endocytic vesicle membrane / serine-type endopeptidase inhibitor activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Vasopressin regulates renal water homeostasis via Aquaporins / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / toxin activity / G alpha (s) signalling events / endosome / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / positive regulation of cell population proliferation / positive regulation of gene expression / perinuclear region of cytoplasm / endoplasmic reticulum / Golgi apparatus / extracellular space / membrane / plasma membrane
Similarity search - Function
Vasopressin V2 receptor / Vasopressin receptor / : / Proteinase inhibitor I2, Kunitz, conserved site / Pancreatic trypsin inhibitor (Kunitz) family signature. / BPTI/Kunitz family of serine protease inhibitors. / Pancreatic trypsin inhibitor Kunitz domain / Kunitz/Bovine pancreatic trypsin inhibitor domain / Pancreatic trypsin inhibitor (Kunitz) family profile. / Pancreatic trypsin inhibitor Kunitz domain superfamily ...Vasopressin V2 receptor / Vasopressin receptor / : / Proteinase inhibitor I2, Kunitz, conserved site / Pancreatic trypsin inhibitor (Kunitz) family signature. / BPTI/Kunitz family of serine protease inhibitors. / Pancreatic trypsin inhibitor Kunitz domain / Kunitz/Bovine pancreatic trypsin inhibitor domain / Pancreatic trypsin inhibitor (Kunitz) family profile. / Pancreatic trypsin inhibitor Kunitz domain superfamily / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Mambaquaretin-1 / Vasopressin V2 receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Dendroaspis angusticeps (eastern green mamba)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsBous, J. / Fouillen, A. / Couvineau, P. / Orcel, H. / Mary, C. / Mendre, C. / Schulte, G. / Granier, S. / Gilles, N. / Mouillac, B.
Funding support France, European Union, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0014 France
Agence Nationale de la Recherche (ANR)ANR-22-CE44-0021 France
H2020 Marie Curie Actions of the European Commission101063049_3D-V2REuropean Union
CitationJournal: Nat Commun / Year: 2025
Title: Inactive structures of the vasopressin V2 receptor reveal distinct binding modes for Tolvaptan and Mambaquaretin toxin.
Authors: Aurélien Fouillen / Julien Bous / Pierre Couvineau / Hélène Orcel / Charline Mary / Lucie Lafleur / Timothé Pierre / Christiane Mendre / Nicolas Gilles / Gunnar Schulte / Sébastien ...Authors: Aurélien Fouillen / Julien Bous / Pierre Couvineau / Hélène Orcel / Charline Mary / Lucie Lafleur / Timothé Pierre / Christiane Mendre / Nicolas Gilles / Gunnar Schulte / Sébastien Granier / Bernard Mouillac /
Abstract: Inhibitors of the arginine-vasopressin (AVP) V2 receptor (V2R) are key therapeutic compounds for treating hyponatremia or polycystic kidney diseases. Rational drug design based on experimental G ...Inhibitors of the arginine-vasopressin (AVP) V2 receptor (V2R) are key therapeutic compounds for treating hyponatremia or polycystic kidney diseases. Rational drug design based on experimental G protein-coupled receptor structures is a powerful avenue to develop better drugs. So far, the lack of inhibitor-bound V2R structures has impaired this strategy. Here we describe the cryo-electron microscopy structures of the V2R in complex with two selective inverse agonists, the non-peptide Tolvaptan (TVP) and the green mamba snake Mambaquaretin toxin (MQ1). Both ligands bind into the orthosteric binding site but with substantial differences. TVP binds deeper than MQ1, and directly contacts the toggle switch residue W284 in the transmembrane domain 6. The Kunitz-fold toxin displays extensive contacts with extracellular and transmembrane residues. As anticipated from TVP and MQ1 pharmacological properties, both structures represent inactive V2R conformations. Their comparison with those of the active AVP-bound V2R reveals the molecular mechanisms modulating receptor activity. The mini-protein MQ1-bound V2R structure suggests a new pharmacology approach for treating water homeostasis and renal diseases.
History
DepositionNov 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mambaquaretin-1
H: Synthetic antibody, anti-BRIL Fab fragment, Heavy chain
K: anti-BRIL Fab Nanobody
L: Synthetic antibody, anti-BRIL Fab fragment, Light chain
R: arginine-vasopressin (AVP) V2 receptor (V2R)-BRIL,Vasopressin V2 receptor,Vasopressin V2 receptor


Theoretical massNumber of molelcules
Total (without water)130,3945
Polymers130,3945
Non-polymers00
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

#1: Protein Mambaquaretin-1 / MQ-1 / MQ1 / Upsilon-Da2a


Mass: 6329.218 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Dendroaspis angusticeps (eastern green mamba)
References: UniProt: A0A1Z0YU59
#2: Antibody Synthetic antibody, anti-BRIL Fab fragment, Heavy chain


Mass: 24539.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody anti-BRIL Fab Nanobody


Mass: 15654.138 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia (bacteria)
#4: Antibody Synthetic antibody, anti-BRIL Fab fragment, Light chain


Mass: 23541.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Protein arginine-vasopressin (AVP) V2 receptor (V2R)-BRIL,Vasopressin V2 receptor,Vasopressin V2 receptor / V2R / AVPR V2 / Antidiuretic hormone receptor / Renal-type arginine vasopressin receptor


Mass: 60330.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AVPR2, ADHR, DIR, DIR3, V2R / Production host: Spodoptera (butterflies/moths) / References: UniProt: P30518
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: Inactive V2R-bril bound to tolvaptan stabillized with anti-BRIL Fab and anti-BRIL Fab Nanobody
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.115 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPU3.6image acquisition
4cryoSPARC4.5.1CTF correction
7Cootmodel fitting
9Rosettamodel refinement
10PHENIXmodel refinement
11cryoSPARC4.5.1initial Euler assignment
12cryoSPARC4.5.1final Euler assignment
13RELION4classification
14cryoSPARC4.5.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202513 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.5 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047299
ELECTRON MICROSCOPYf_angle_d0.6459936
ELECTRON MICROSCOPYf_dihedral_angle_d10.9532505
ELECTRON MICROSCOPYf_chiral_restr0.0411133
ELECTRON MICROSCOPYf_plane_restr0.0051258

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