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- PDB-8th4: Structure of AT118-L Nanobody Antagonist in Complex with the Angi... -

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Basic information

Entry
Database: PDB / ID: 8th4
TitleStructure of AT118-L Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor and Losartan
Components
  • AT118-L Nanobody
  • BAG2 Anti-BRIL Fab Heavy Chain
  • BAG2 Anti-BRIL Fab Light Chain
  • Type-1 angiotensin II receptor Soluble cytochrome b562 complex
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 / positive regulation of phospholipase A2 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 macrophage derived foam cell differentiation ...angiotensin type I receptor activity / positive regulation of phospholipase A2 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 macrophage derived foam cell differentiation / positive regulation of CoA-transferase activity / low-density lipoprotein particle remodeling / regulation of systemic arterial blood pressure by renin-angiotensin / Rho protein signal transduction / regulation of vasoconstriction / positive regulation of protein metabolic process / blood vessel diameter maintenance / Peptide ligand-binding receptors / cell chemotaxis / kidney development / angiotensin-activated signaling pathway / regulation of cell growth / calcium-mediated signaling / electron transport chain / positive regulation of inflammatory response / positive regulation of reactive oxygen species metabolic process / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / regulation of cell population proliferation / phospholipase C-activating G protein-coupled receptor signaling pathway / regulation of inflammatory response / positive regulation of cytosolic calcium ion concentration / G alpha (q) signalling events / periplasmic space / electron transfer activity / inflammatory response / iron ion binding / symbiont entry into host cell / protein heterodimerization activity / G protein-coupled receptor signaling pathway / heme binding / 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
Chem-LSN / Soluble cytochrome b562 / Type-1 angiotensin II receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)113,3505
Polymers112,9274
Non-polymers4231
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 Type-1 angiotensin II receptor Soluble cytochrome b562 complex / AT1AR / AT1BR / Angiotensin II type-1 receptor / AT1 receptor / Cytochrome b-562


Mass: 49501.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (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) / 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) / Production host: Homo sapiens (human)
#4: Antibody AT118-L Nanobody


Mass: 15345.063 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#5: Chemical ChemComp-LSN / [2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol / Losartan


Mass: 422.911 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H23ClN6O / Comment: medication*YM
Has ligand of interestN

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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-L Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion protein, Anti-BRIL Fab, Anti-Fab Nanobody, and Losartan
Type: COMPLEX / Entity ID: #1-#4 / 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.: 7 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: 64.1 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
9cryoSPARC3initial Euler assignment
10cryoSPARC3final Euler assignment
12cryoSPARC33D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 405950
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238621 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 83.12 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315841
ELECTRON MICROSCOPYf_angle_d0.53167932
ELECTRON MICROSCOPYf_chiral_restr0.0384889
ELECTRON MICROSCOPYf_plane_restr0.0047993
ELECTRON MICROSCOPYf_dihedral_angle_d3.846819

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