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- PDB-9eah: Structure of nanobody AT209 in complex with the olmesartan-bound ... -

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Basic information

Entry
Database: PDB / ID: 9eah
TitleStructure of nanobody AT209 in complex with the olmesartan-bound angiotensin II type I receptor (AT1R)
Components
  • BAG2 Anti-BRIL Fab Heavy Chain
  • BAG2 Anti-BRIL Fab Light Chain
  • Nanobody AT209,Type-1 angiotensin II receptor,Soluble cytochrome b562
KeywordsMEMBRANE PROTEIN / GPCR / AT1R / nanobody
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
CHOLESTEROL / Olmesartan / 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: 3.1 Å
AuthorsSkiba, M.A. / Kruse, A.C.
Funding support United States, 4items
OrganizationGrant numberCountry
Helen Hay Whitney Foundation United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)K99HD110612 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)R21HD101596 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA260415 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Epitope-directed selection of GPCR nanobody ligands with evolvable function.
Authors: Meredith A Skiba / Clare Canavan / Genevieve R Nemeth / Jinghan Liu / Ali Kanso / Andrew C Kruse /
Abstract: Antibodies have the potential to target G protein-coupled receptors (GPCRs) with high receptor, cellular, and tissue selectivity; however, few antibody ligands for GPCRs exist. Here, we describe a ...Antibodies have the potential to target G protein-coupled receptors (GPCRs) with high receptor, cellular, and tissue selectivity; however, few antibody ligands for GPCRs exist. Here, we describe a generalizable selection method to enrich for GPCR ligands from a synthetic camelid antibody fragment (nanobody) library. Our strategy yielded multiple nanobody ligands for the angiotensin II type I receptor (AT1R), a prototypical GPCR and important drug target. We found that nanobodies readily act as allosteric modulators, encoding selectivity for both the receptor and chemical features of GPCR ligands. We then used structure-guided design to convert two nanobodies from allosteric ligands to competitive AT1R inhibitors through simple mutations. This work demonstrates that nanobodies can encode multiple pharmacological behaviors and have great potential as evolvable scaffolds for the development of next-generation GPCR therapeutics.
History
DepositionNov 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
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Revision 1.1Mar 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nanobody AT209,Type-1 angiotensin II receptor,Soluble cytochrome b562
C: BAG2 Anti-BRIL Fab Heavy Chain
D: BAG2 Anti-BRIL Fab Light Chain
B: Nanobody AT209,Type-1 angiotensin II receptor,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,2776
Polymers178,4434
Non-polymers8332
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 Nanobody AT209,Type-1 angiotensin II receptor,Soluble cytochrome b562 / AT1AR / AT1BR / Angiotensin II type-1 receptor / AT1 receptor / Cytochrome b-562


Mass: 65181.449 Da / Num. of mol.: 2 / Mutation: M232W, H327I, R331L
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 line (production host): Expi293R / 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 line (production host): Expi293R / 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 line (production host): Expi293R / Production host: Homo sapiens (human)
#4: Chemical ChemComp-OLM / Olmesartan


Mass: 446.502 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H26N6O3 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
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: nanobody AT209 in complex with the olmesartan-bound angiotensin II type I receptor (AT1R)
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) / Strain: Expi293R
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 903168
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251356 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025751
ELECTRON MICROSCOPYf_angle_d0.4347826
ELECTRON MICROSCOPYf_dihedral_angle_d12.2032061
ELECTRON MICROSCOPYf_chiral_restr0.038881
ELECTRON MICROSCOPYf_plane_restr0.003959

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