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- PDB-7qsr: CryoEM structure of the Ectodomain of Human PLA2R -

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Basic information

Entry
Database: PDB / ID: 7qsr
TitleCryoEM structure of the Ectodomain of Human PLA2R
ComponentsSecretory phospholipase A2 receptor
KeywordsMEMBRANE PROTEIN / Receptor / Ectodomain / Glycoprotein / Nephrology
Function / homology
Function and homology information


negative regulation of arachidonate secretion / negative regulation of phospholipase A2 activity / Acyl chain remodelling of PG / Acyl chain remodelling of PC / Acyl chain remodelling of PI / positive regulation of DNA damage response, signal transduction by p53 class mediator / Acyl chain remodelling of PS / Acyl chain remodelling of PE / Synthesis of PA / positive regulation of podocyte apoptotic process ...negative regulation of arachidonate secretion / negative regulation of phospholipase A2 activity / Acyl chain remodelling of PG / Acyl chain remodelling of PC / Acyl chain remodelling of PI / positive regulation of DNA damage response, signal transduction by p53 class mediator / Acyl chain remodelling of PS / Acyl chain remodelling of PE / Synthesis of PA / positive regulation of podocyte apoptotic process / positive regulation of arachidonate secretion / oxidative stress-induced premature senescence / phospholipase binding / replicative senescence / reactive oxygen species metabolic process / receptor-mediated endocytosis / positive regulation of cytokine production / signaling receptor activity / carbohydrate binding / receptor complex / cell surface / extracellular region / plasma membrane
Similarity search - Function
Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. / Fibronectin type-II collagen-binding domain profile. / Fibronectin type 2 domain / C-type lectin, conserved site / C-type lectin domain signature. / Ricin-type beta-trefoil / Lectin domain of ricin B chain profile. ...Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. / Fibronectin type-II collagen-binding domain profile. / Fibronectin type 2 domain / C-type lectin, conserved site / C-type lectin domain signature. / Ricin-type beta-trefoil / Lectin domain of ricin B chain profile. / Ricin B, lectin domain / Ricin B-like lectins / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / Lectin C-type domain / C-type lectin-like/link domain superfamily / C-type lectin fold
Similarity search - Domain/homology
Secretory phospholipase A2 receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBriggs, D.C. / Lockhart-Cairns, M.P. / Baldock, C.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
European Union (EU)305608 United Kingdom
Wellcome Trust097820 United Kingdom
Wellcome Trust203128/Z/16/Z United Kingdom
Wellcome Trust202860/Z/16/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structure of PLA2R reveals presentation of the dominant membranous nephropathy epitope and an immunogenic patch.
Authors: Maryline Fresquet / Michael P Lockhart-Cairns / Samuel J Rhoden / Thomas A Jowitt / David C Briggs / Clair Baldock / Paul E Brenchley / Rachel Lennon /
Abstract: Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most ...Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.
History
DepositionJan 14, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Secretory phospholipase A2 receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,95014
Polymers159,2881
Non-polymers4,66113
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, See related entries
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4900 Å2
ΔGint63 kcal/mol
Surface area62650 Å2

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Components

#1: Protein Secretory phospholipase A2 receptor / PLA2-R / PLA2R / 180 kDa secretory phospholipase A2 receptor / C-type lectin domain family 13 ...PLA2-R / PLA2R / 180 kDa secretory phospholipase A2 receptor / C-type lectin domain family 13 member C / M-type receptor


Mass: 159288.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Extracellular domain of Human PLA2R with C-terminal His tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: PLA2R1, CLEC13C / Cell line (production host): HEK293-EBNA1 / Production host: Homo sapiens (human) / References: UniProt: Q13018
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#4: Polysaccharide alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-6)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpb1-3DGlcpNAcb1-6DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a6-b1_b3-c1_c6-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(6+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human PLA2R Ectodomain / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK-EBNA1
Buffer solutionpH: 6.2 / Details: PLA2R prepared in Bis-Tris pH 6.2 buffer
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11COPPER300C-flat-2/2
21COPPER300Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Cryogen: NITROGEN / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDDetails
1
230 degree Tilt dataset
Image recording
IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Film or detector modelNum. of real images
11214.8GATAN K3 (6k x 4k)3702
22214.2GATAN K3 (6k x 4k)2137
EM imaging optics
Energyfilter nameIDImaging-IDEnergyfilter slit width (eV)
GIF Quantum ER1120
GIF Quantum ER2220

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameVersionCategory
7PHENIX1.19.2model fitting
10PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1700000
Details: Particles were picked using a Gaussian blob (80-130 ang) from 100 images of the 0 deg data set were extracted in a 256 pixel box, binned 4x4 (64 pixel box), and subjected to 2D ...Details: Particles were picked using a Gaussian blob (80-130 ang) from 100 images of the 0 deg data set were extracted in a 256 pixel box, binned 4x4 (64 pixel box), and subjected to 2D classification. The best templates were used to template pick all images from both data sets.
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 221000 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient
Atomic model buildingPDB-ID: 7JPT

7jpt


Pdb chain-ID: A / Pdb chain residue range: 30-1381
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 186.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003111539
ELECTRON MICROSCOPYf_angle_d0.62815673
ELECTRON MICROSCOPYf_chiral_restr0.04281666
ELECTRON MICROSCOPYf_plane_restr0.00451975
ELECTRON MICROSCOPYf_dihedral_angle_d6.58731587

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