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- EMDB-41248: Structure of AT118-H Nanobody Antagonist in Complex with the Angi... -

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Basic information

Entry
Database: EMDB / ID: EMD-41248
TitleStructure of AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor
Map dataLocally Refined Sharpened Map of AT118-H, AT1R-BRIL and BAG2 Anti-BRIL Fab Variable Domains
Sample
  • Complex: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion, Anti-BRIL Fab, and Anti-Fab Nanobody
    • Protein or peptide: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
    • Protein or peptide: BAG2 Anti-BRIL Fab Heavy Chain
    • Protein or peptide: BAG2 Anti-BRIL Fab Light Chain
  • Ligand: CHOLESTEROL HEMISUCCINATE
KeywordsG 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 / : ...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 / : / low-density lipoprotein particle remodeling / positive regulation of macrophage derived foam cell differentiation / positive regulation of protein metabolic process / regulation of systemic arterial blood pressure by renin-angiotensin / Rho protein signal transduction / regulation of vasoconstriction / Peptide ligand-binding receptors / blood vessel diameter maintenance / kidney development / angiotensin-activated signaling pathway / cell chemotaxis / 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 / 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 / inflammatory response / iron ion binding / protein heterodimerization activity / symbiont entry into host cell / 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
Soluble cytochrome b562 / Type-1 angiotensin II receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSkiba MA / Kruse AC
Funding support United States, 7 items
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, 2023-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41248.png

Downloads & links

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Map

FileDownload / File: emd_41248.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally Refined Sharpened Map of AT118-H, AT1R-BRIL and BAG2 Anti-BRIL Fab Variable Domains
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.398
Minimum - Maximum-1.6356825 - 2.244838
Average (Standard dev.)0.0010048223 (±0.040248457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Half Map A of Global Map of AT118-H...

Fileemd_41248_additional_1.map
AnnotationHalf Map A of Global Map of AT118-H and AT1R-BRIL in complex with BAG2 Anti-BRIL Fab and Anti-Fab Nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half Map B of Global Map of AT118-H...

Fileemd_41248_additional_2.map
AnnotationHalf Map B of Global Map of AT118-H and AT1R-BRIL in complex with BAG2 Anti-BRIL Fab and Anti-Fab Nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Global Sharpened Map of AT118-H and AT1R-BRIL in...

Fileemd_41248_additional_3.map
AnnotationGlobal Sharpened Map of AT118-H and AT1R-BRIL in complex with BAG2 Anti-BRIL Fab and Anti-Fab Nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B of Locally Refined Sharpened Map...

Fileemd_41248_half_map_1.map
AnnotationHalf Map B of Locally Refined Sharpened Map of AT118-H, AT1R-BRIL and BAG2 Anti-BRIL Fab Variable Domains
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A of Locally Refined Sharpened Map...

Fileemd_41248_half_map_2.map
AnnotationHalf Map A of Locally Refined Sharpened Map of AT118-H, AT1R-BRIL and BAG2 Anti-BRIL Fab Variable Domains
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AT118-H Nanobody Antagonist in Complex with the Angiotensin II Ty...

EntireName: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion, Anti-BRIL Fab, and Anti-Fab Nanobody
Components
  • Complex: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion, Anti-BRIL Fab, and Anti-Fab Nanobody
    • Protein or peptide: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
    • Protein or peptide: BAG2 Anti-BRIL Fab Heavy Chain
    • Protein or peptide: BAG2 Anti-BRIL Fab Light Chain
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Ty...

SupramoleculeName: AT118-H Nanobody Antagonist in Complex with the Angiotensin II Type I Receptor BRIL Fusion, Anti-BRIL Fab, and Anti-Fab Nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochr...

MacromoleculeName: AT118-H nanobody, Type-1 angiotensin II receptor, Soluble cytochrome b562 complex
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.364684 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGYIYR RYRMGWYRQA PGKGREFVAA ISGGSSTNYA DSVKGRFTIS RDNSKNTVYL QMNSLRAED TAVYYCAAYR IVSDPRVYWG QGTQVTVSSL EGGSGEDQVD PRLIDGKILN SSTEDGIKRI QDDCPKAGRH N YIFVMIPT ...String:
EVQLVESGGG LVQPGGSLRL SCAASGYIYR RYRMGWYRQA PGKGREFVAA ISGGSSTNYA DSVKGRFTIS RDNSKNTVYL QMNSLRAED TAVYYCAAYR IVSDPRVYWG QGTQVTVSSL EGGSGEDQVD PRLIDGKILN SSTEDGIKRI QDDCPKAGRH N YIFVMIPT LYSIIFVVGI FGNSLVVIVI YFYMKLKTVA SVFLLNLALA DLCFLLTLPL WAVYTAMEYR WPFGNYLCKI AS ASVSFNL YASVFLLTCL SIDRYLAIVH PMKSRLRRTM LVAKVTCIII WLLAGLASLP AIIHRNVFFI ENTNITVCAF HYE SQNSTL PIGLGLTKNI LGFLFPFLII LTSYTLIWKA LKKAYDLEDN WETLNDNLKV IEKADNAAQV KDALTKMRAA ALDA QKATP PKLEDKSPDS PEMKDFRHGF DILVGQIDDA LKLANEGKVK EAQAAAEQLK TTRNAYIQKY LERARSTLDK LNDDI FKII MAIVLFFFFS WIPHQIFTFL DVLIQLGIIR DCRIADIVDT AMPITICIAY FNNCLNPLFY GFLGKKFKRY FLQLLK YGG SSLEVLFQGP TETSQVAPA

UniProtKB: Type-1 angiotensin II receptor, Soluble cytochrome b562, Type-1 angiotensin II receptor

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Macromolecule #2: BAG2 Anti-BRIL Fab Heavy Chain

MacromoleculeName: BAG2 Anti-BRIL Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.539314 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCD

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Macromolecule #3: BAG2 Anti-BRIL Fab Light Chain

MacromoleculeName: BAG2 Anti-BRIL Fab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.541164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration9.2 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% GDN, 0.005% CHS
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
SoftwareName: SerialEM (ver. 4.0.5)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 64.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6309448
Startup modelType of model: OTHER / Details: Ab Initio Model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 478571
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)

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