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- PDB-9eai: Structure of nanobody AT206 in complex with the losartan-bound an... -

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Basic information

Entry
Database: PDB / ID: 9eai
TitleStructure of nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
Components
  • (BAG2 Anti-BRIL Fab ...) x 2
  • Nanobody AT206,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 / Chem-LSN / 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: 3.1 Å
AuthorsSkiba, M.A. / Liu, J. / 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
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562
B: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562
C: BAG2 Anti-BRIL Fab Heavy Chain
D: BAG2 Anti-BRIL Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,9498
Polymers179,1664
Non-polymers1,7834
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

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Protein , 1 types, 2 molecules AB

#1: Protein Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562 / AT1AR / AT1BR / Angiotensin II type-1 receptor / AT1 receptor / Cytochrome b-562


Mass: 65542.836 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 / Production host: Homo sapiens (human) / Strain (production host): Expi293F / References: UniProt: P30556, UniProt: P0ABE7

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Antibody , 2 types, 2 molecules CD

#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) / Strain (production host): Expi293F
#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) / Strain (production host): Expi293F

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Non-polymers , 3 types, 4 molecules

#4: 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 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#5: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4
#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O

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Details

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 AT206 in complex with the losartan-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 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4322

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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: 1748565
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 379250 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035735
ELECTRON MICROSCOPYf_angle_d0.4637815
ELECTRON MICROSCOPYf_dihedral_angle_d10.6032220
ELECTRON MICROSCOPYf_chiral_restr0.038894
ELECTRON MICROSCOPYf_plane_restr0.003938

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