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- EMDB-42185: Cryo-EM structure of POmAb, a Type-I anti-prothrombin antiphospho... -

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Entry
Database: EMDB / ID: EMD-42185
TitleCryo-EM structure of POmAb, a Type-I anti-prothrombin antiphospholipid antibody, bound to kringle-1 of human prothrombin
Map data
Sample
  • Complex: Complex of Fab fragment of POmAb with human prothrombin
    • Protein or peptide: POmAb Light Chain
    • Protein or peptide: POmAb Heavy Chain
    • Protein or peptide: ProthrombinThrombin
KeywordsAutoimmunity / Thrombosis / Complex / Immunoglobulin / Coagulation factor / Anticoagulation / Inhibitor / BLOOD CLOTTING
Function / homology
Function and homology information


positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin ...positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / negative regulation of astrocyte differentiation / negative regulation of platelet activation / positive regulation of collagen biosynthetic process / negative regulation of cytokine production involved in inflammatory response / positive regulation of blood coagulation / negative regulation of fibrinolysis / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / regulation of cytosolic calcium ion concentration / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / Regulation of Complement cascade / acute-phase response / Cell surface interactions at the vascular wall / lipopolysaccharide binding / negative regulation of proteolysis / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / positive regulation of insulin secretion / platelet activation / response to wounding / Golgi lumen / positive regulation of protein localization to nucleus / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / positive regulation of reactive oxygen species metabolic process / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / Thrombin signalling through proteinase activated receptors (PARs) / heparin binding / regulation of cell shape / positive regulation of cell growth / G alpha (q) signalling events / collagen-containing extracellular matrix / blood microparticle / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / calcium ion binding / positive regulation of cell population proliferation / proteolysis / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. ...Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / Kringle-like fold / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKumar S / Summers B / Basore K / Pozzi N
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL150146 United States
National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS)UL1 TR002345 United States
CitationJournal: Blood / Year: 2024
Title: Cryo-EM structure and functional basis of prothrombin recognition by a Type-I anti-prothrombin antiphospholipid antibody.
Authors: Suresh Kumar / Brock Summers / Kathrine Basore / Vittorio Pengo / Robert Flaumenhaft / Nicola Pozzi /
Abstract: Anti-prothrombin (anti-PT) antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered ...Anti-prothrombin (anti-PT) antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered Type-I and Type-II antibodies and proposed that Type-I recognize the open form. In this study, we report the discovery, structural and functional characterization in human plasma of a Type-I antibody, POmAb. Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium towards the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russel Viper Venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a Type-I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.
History
DepositionOct 3, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42185.png

Downloads & links

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Map

FileDownload / File: emd_42185.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.081 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.0016774293 - 2.0759661
Average (Standard dev.)0.00041560567 (±0.01552948)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_42185_additional_1.map
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Additional map: #1

Fileemd_42185_additional_2.map
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Half map: #2

Fileemd_42185_half_map_1.map
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Half map: #1

Fileemd_42185_half_map_2.map
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Sample components

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Entire : Complex of Fab fragment of POmAb with human prothrombin

EntireName: Complex of Fab fragment of POmAb with human prothrombin
Components
  • Complex: Complex of Fab fragment of POmAb with human prothrombin
    • Protein or peptide: POmAb Light Chain
    • Protein or peptide: POmAb Heavy Chain
    • Protein or peptide: ProthrombinThrombin

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Supramolecule #1: Complex of Fab fragment of POmAb with human prothrombin

SupramoleculeName: Complex of Fab fragment of POmAb with human prothrombin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragment generated by proteolytic cleavage of POmAb IgG antibody in complex with open conformation of human prothrombin
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: POmAb Light Chain

MacromoleculeName: POmAb Light Chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.558154 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QIVLTQSPAI MSVSLGERVT MTCIVSSSVS STYLHWYQQK PGSSPKLWIY SSSNLASGVP TRFSGSGSGT SHSLTISSME AEDAAAYYC QLYRRSPLTF GAGTKLELKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
QIVLTQSPAI MSVSLGERVT MTCIVSSSVS STYLHWYQQK PGSSPKLWIY SSSNLASGVP TRFSGSGSGT SHSLTISSME AEDAAAYYC QLYRRSPLTF GAGTKLELKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC

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Macromolecule #2: POmAb Heavy Chain

MacromoleculeName: POmAb Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.406252 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVHLVESGGG LVKPGGSLKL SCAASGFTFS SYYMYWVRQT PEKRLEWVAT ISNGGIYTYY LDSVRGRFTI SRDNAKNILY LQMSGLSSA DSAIYYCTRD GERQGAMDYW GQGTSVTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG ...String:
EVHLVESGGG LVKPGGSLKL SCAASGFTFS SYYMYWVRQT PEKRLEWVAT ISNGGIYTYY LDSVRGRFTI SRDNAKNILY LQMSGLSSA DSAIYYCTRD GERQGAMDYW GQGTSVTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VPSSTWPSET VTCNVAHPAS STKVDKKIVP

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Macromolecule #3: Prothrombin

MacromoleculeName: Prothrombin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.386113 KDa
SequenceString: ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVF WAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNIT RSGIECQLW RSRYPHKPEI NSTTHPGADL QENFCRNPDS STTGPWCYTT DPTVRRQECS IPVCGQDQVT VAMTPRSEGS S VNLSPPLE ...String:
ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVF WAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNIT RSGIECQLW RSRYPHKPEI NSTTHPGADL QENFCRNPDS STTGPWCYTT DPTVRRQECS IPVCGQDQVT VAMTPRSEGS S VNLSPPLE QCVPDRGQQY QGRLAVTTHG LPCLAWASAQ AKALSKHQDF NSAVQLVENF CRNPDGDEEG VWCYVAGKPG DF GYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYI DGR IVEGSD AEIGMSPWQV MLFRKSPQEL LCGASLISDR WVLTAAHCLL YPPWDKNFTE NDLLVRIGKH SRTRYERNIE KISM LEKIY IHPRYNWREN LDRDIALMKL KKPVAFSDYI HPVCLPDRET AASLLQAGYK GRVTGWGNLK ETWTANVGKG QPSVL QVVN LPIVERPVCK DSTRIRITDN MFCAGYKPDE GKRGDACEGD SGGPFVMKSP FNNRWYQMGI VSWGEGCDRD GKYGFY THV FRLKKWIQKV IDQFGE

UniProtKB: Prothrombin

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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

Concentration0.33 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse particles. Complex purified by SEC.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Specialist opticsSpherical aberration corrector: Microscope is outfitted with a Cs image corrector with two hexapole elements.
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 82.0 K / Max: 84.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 62.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 176744
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8uf7:
Cryo-EM structure of POmAb, a Type-I anti-prothrombin antiphospholipid antibody, bound to kringle-1 of human prothrombin

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