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- PDB-8th3: Structure of AT118-H Nanobody Antagonist in Complex with the Angi... -

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Basic information

Entry
Database: PDB / ID: 8th3
TitleStructure of AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor
Components
  • AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
  • BAG2 Anti-BRIL Fab Heavy Chain
  • BAG2 Anti-BRIL Fab Light Chain
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / G protein-coupled receptor / nanobody / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


angiotensin type I receptor activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of cholesterol metabolic process / low-density lipoprotein particle remodeling ...angiotensin type I receptor activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of cholesterol metabolic process / low-density lipoprotein particle remodeling / positive regulation of macrophage derived foam cell differentiation / regulation of systemic arterial blood pressure by renin-angiotensin / regulation of vasoconstriction / Rho protein signal transduction / Peptide ligand-binding receptors / blood vessel diameter maintenance / angiotensin-activated signaling pathway / cell chemotaxis / kidney development / regulation of cell growth / calcium-mediated signaling / electron transport chain / positive regulation of reactive oxygen species metabolic process / positive regulation of inflammatory response / Cargo recognition for clathrin-mediated endocytosis / regulation of cell population proliferation / Clathrin-mediated endocytosis / positive regulation of cytosolic calcium ion concentration / regulation of inflammatory response / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / iron ion binding / inflammatory response / protein heterodimerization activity / heme binding / symbiont entry into host cell / membrane / plasma membrane
Similarity search - Function
Angiotensin II receptor type 1 / Angiotensin II receptor family / : / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM ...Angiotensin II receptor type 1 / Angiotensin II receptor family / : / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / 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
CHOLESTEROL HEMISUCCINATE / Soluble cytochrome b562 / Type-1 angiotensin II receptor
Similarity search - Component
Biological speciesCamelidae (mammal)
Homo sapiens (human)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsSkiba, M.A. / Kruse, A.C.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorOD021345 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)HD101596 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA260415 United States
The Vallee Foundation Inc. United States
Richard and Susan Smith Family Foundation United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS088566 United States
The Pew Charitable Trusts United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Antibodies expand the scope of angiotensin receptor pharmacology.
Authors: Meredith A Skiba / Sarah M Sterling / Shaun Rawson / Shuhao Zhang / Huixin Xu / Haoran Jiang / Genevieve R Nemeth / Morgan S A Gilman / Joseph D Hurley / Pengxiang Shen / Dean P Staus / ...Authors: Meredith A Skiba / Sarah M Sterling / Shaun Rawson / Shuhao Zhang / Huixin Xu / Haoran Jiang / Genevieve R Nemeth / Morgan S A Gilman / Joseph D Hurley / Pengxiang Shen / Dean P Staus / Jihee Kim / Conor McMahon / Maria K Lehtinen / Howard A Rockman / Patrick Barth / Laura M Wingler / Andrew C Kruse /
Abstract: G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to ...G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.
History
DepositionJul 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Dec 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
B: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
C: BAG2 Anti-BRIL Fab Heavy Chain
D: BAG2 Anti-BRIL Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,7836
Polymers178,8104
Non-polymers9732
Water00
1


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

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Components

#1: Protein AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex / AT1AR / AT1BR / Angiotensin II type-1 receptor / AT1 receptor / Cytochrome b-562


Mass: 65364.684 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae (mammal), (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: AGTR1, AGTR1A, AGTR1B, AT2R1, AT2R1B, cybC / Cell (production host): Expi293 Tet-R / Production host: Homo sapiens (human) / References: UniProt: P30556, UniProt: P0ABE7
#2: Antibody BAG2 Anti-BRIL Fab Heavy Chain


Mass: 24539.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Cell (production host): Expi293 Tet-R / Production host: Homo sapiens (human)
#3: Antibody BAG2 Anti-BRIL Fab Light Chain


Mass: 23541.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Cell (production host): Expi293 Tet-R / Production host: Homo sapiens (human)
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestN
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: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion, Anti-BRIL Fab, and Anti-Fab Nanobody
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293 Tet-R
Buffer solutionpH: 7.4
Details: 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% GDN, 0.005% CHS
SpecimenConc.: 9.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: 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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2SerialEM4.0.5image acquisition
4cryoSPARC3CTF correction
9PHENIX1.20.1model refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6309448
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 478571 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 60.2 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325913
ELECTRON MICROSCOPYf_angle_d0.51938037
ELECTRON MICROSCOPYf_chiral_restr0.0389909
ELECTRON MICROSCOPYf_plane_restr0.0043986
ELECTRON MICROSCOPYf_dihedral_angle_d4.1826818

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