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- PDB-8xvj: Cryo-EM structure of ETAR bound with Macitentan -

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Basic information

Entry
Database: PDB / ID: 8xvj
TitleCryo-EM structure of ETAR bound with Macitentan
Components
  • Endoglucanase H,Endothelin-1 receptor,Soluble cytochrome b562
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
  • anti-Fab Nanobody
KeywordsSIGNALING PROTEIN / GPCR / COMPLEX / ETA / MACITENTAN
Function / homology
Function and homology information


regulation of protein localization to cell leading edge / endothelin receptor activity / cellular response to human chorionic gonadotropin stimulus / meiotic cell cycle process involved in oocyte maturation / semaphorin-plexin signaling pathway involved in axon guidance / neural crest cell fate commitment / atrial cardiac muscle tissue development / glomerular endothelium development / sympathetic neuron axon guidance / vascular associated smooth muscle cell development ...regulation of protein localization to cell leading edge / endothelin receptor activity / cellular response to human chorionic gonadotropin stimulus / meiotic cell cycle process involved in oocyte maturation / semaphorin-plexin signaling pathway involved in axon guidance / neural crest cell fate commitment / atrial cardiac muscle tissue development / glomerular endothelium development / sympathetic neuron axon guidance / vascular associated smooth muscle cell development / noradrenergic neuron differentiation / cardiac chamber formation / heparin metabolic process / developmental pigmentation / pharyngeal arch artery morphogenesis / regulation of D-glucose transmembrane transport / endothelin receptor signaling pathway involved in heart process / cardiac neural crest cell migration involved in outflow tract morphogenesis / endothelin receptor signaling pathway / response to acetylcholine / sodium ion homeostasis / podocyte differentiation / podocyte apoptotic process / renal sodium ion absorption / embryonic skeletal system development / left ventricular cardiac muscle tissue morphogenesis / artery smooth muscle contraction / mesenchymal cell apoptotic process / glomerular filtration / protein transmembrane transport / enteric nervous system development / axonogenesis involved in innervation / positive regulation of cation channel activity / cellular response to follicle-stimulating hormone stimulus / cranial skeletal system development / cellular response to luteinizing hormone stimulus / renal albumin absorption / respiratory gaseous exchange by respiratory system / sympathetic nervous system development / phosphatidylinositol phospholipase C activity / norepinephrine metabolic process / vasoconstriction / embryonic heart tube development / axon extension / establishment of endothelial barrier / aorta development / middle ear morphogenesis / cellulase / neuromuscular process / beta-glucosidase activity / cellulase activity / neuron remodeling / branching involved in blood vessel morphogenesis / face development / thyroid gland development / smooth muscle contraction / blood vessel remodeling / canonical Wnt signaling pathway / cellulose catabolic process / activation of adenylate cyclase activity / protein kinase A signaling / response to amphetamine / regulation of heart rate / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / mitochondrion organization / calcium ion transmembrane transport / electron transport chain / regulation of blood pressure / response to wounding / intracellular calcium ion homeostasis / mitotic cell cycle / phospholipase C-activating G protein-coupled receptor signaling pathway / cellular response to oxidative stress / positive regulation of cytosolic calcium ion concentration / gene expression / G alpha (q) signalling events / positive regulation of canonical NF-kappaB signal transduction / in utero embryonic development / cell population proliferation / periplasmic space / electron transfer activity / response to hypoxia / iron ion binding / G protein-coupled receptor signaling pathway / heme binding / cell surface / signal transduction / extracellular region / plasma membrane
Similarity search - Function
Endothelin receptor A / Endothelin receptor family / : / Carbohydrate binding module family 11 / Carbohydrate binding domain (family 11) / Glycosyl hydrolase family 26 / Glycosyl hydrolase family 26 domain / Glycosyl hydrolases family 26 (GH26) domain profile. / : / Clostridium cellulosome enzymes repeated domain signature. ...Endothelin receptor A / Endothelin receptor family / : / Carbohydrate binding module family 11 / Carbohydrate binding domain (family 11) / Glycosyl hydrolase family 26 / Glycosyl hydrolase family 26 domain / Glycosyl hydrolases family 26 (GH26) domain profile. / : / Clostridium cellulosome enzymes repeated domain signature. / Glycoside hydrolase, family 5, conserved site / Glycosyl hydrolases family 5 signature. / Dockerin domain / Dockerin domain profile. / Dockerin type I domain / Dockerin type I repeat / Dockerin domain superfamily / Glycoside hydrolase, family 5 / Cellulase (glycosyl hydrolase family 5) / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Galactose-binding-like domain superfamily / EF-hand calcium-binding domain. / 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) / Glycoside hydrolase superfamily
Similarity search - Domain/homology
: / Soluble cytochrome b562 / Endoglucanase H / Endothelin-1 receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
Acetivibrio thermocellus (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsHou, J.Y. / Liu, S.H. / Wu, L.J. / Liu, Z.J. / Hua, T.
Funding support China, 1items
OrganizationGrant numberCountry
Not funded China
CitationJournal: Cell Discov / Year: 2024
Title: Structural basis of antagonist selectivity in endothelin receptors.
Authors: Junyi Hou / Shenhui Liu / Xiaodan Zhang / Guowei Tu / Lijie Wu / Yijie Zhang / Hao Yang / Xiangcheng Li / Junlin Liu / Longquan Jiang / Qiwen Tan / Fang Bai / Zhijie Liu / Changhong Miao / Tian Hua / Zhe Luo /
Abstract: Endothelins and their receptors, ET and ET, play vital roles in maintaining vascular homeostasis. Therapeutically targeting endothelin receptors, particularly through ET antagonists, has shown ...Endothelins and their receptors, ET and ET, play vital roles in maintaining vascular homeostasis. Therapeutically targeting endothelin receptors, particularly through ET antagonists, has shown efficacy in treating pulmonary arterial hypertension (PAH) and other cardiovascular- and renal-related diseases. Here we present cryo-electron microscopy structures of ET in complex with two PAH drugs, macitentan and ambrisentan, along with zibotentan, a selective ET antagonist, respectively. Notably, a specialized anti-ET antibody facilitated the structural elucidation. These structures, together with the active-state structures of ET-1-bound ET and ET, and the agonist BQ3020-bound ET, in complex with G, unveil the molecular basis of agonist/antagonist binding modes in endothelin receptors. Key residues that confer antagonist selectivity to endothelin receptors were identified along with the activation mechanism of ET. Furthermore, our results suggest that ECL2 in ET can serve as an epitope for antibody-mediated receptor antagonism. Collectively, these insights establish a robust theoretical framework for the rational design of small-molecule drugs and antibodies with selective activity against endothelin receptors.
History
DepositionJan 15, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: anti-BRIL Fab Heavy chain
K: anti-Fab Nanobody
L: anti-BRIL Fab Light chain
R: Endoglucanase H,Endothelin-1 receptor,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,9045
Polymers158,3164
Non-polymers5881
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: Antibody anti-BRIL Fab Heavy chain


Mass: 28577.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
#2: Antibody anti-Fab Nanobody


Mass: 15071.431 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody anti-BRIL Fab Light chain


Mass: 25575.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
#4: Protein Endoglucanase H,Endothelin-1 receptor,Soluble cytochrome b562 / Cellulase H / Endo-1 / 4-beta-glucanase H / EgH / Endothelin receptor type A / ET-A / ETA-R / hET- ...Cellulase H / Endo-1 / 4-beta-glucanase H / EgH / Endothelin receptor type A / ET-A / ETA-R / hET-AR / Cytochrome b-562


Mass: 89090.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) (bacteria), (gene. exp.) Homo sapiens (human), (gene. exp.) ...Source: (gene. exp.) Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) (bacteria), (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: celH, Cthe_1472, EDNRA, ETA, ETRA, cybC / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P16218, UniProt: P25101, UniProt: P0ABE7, cellulase
#5: Chemical ChemComp-A1D5I / 6-[2-(5-bromanylpyrimidin-2-yl)oxyethoxy]-5-(4-bromophenyl)-~{N}-(propylsulfamoyl)pyrimidin-4-amine / Macitentan


Mass: 588.273 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H20Br2N6O4S / Feature type: SUBJECT OF INVESTIGATION
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: Complex of Endothelin receptor type A with macitentan / Type: COMPLEX / Entity ID: #1, #3-#4, #2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 568027 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037600
ELECTRON MICROSCOPYf_angle_d0.6310318
ELECTRON MICROSCOPYf_dihedral_angle_d4.8191058
ELECTRON MICROSCOPYf_chiral_restr0.041161
ELECTRON MICROSCOPYf_plane_restr0.0051301

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