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Yorodumi- PDB-8uf7: Cryo-EM structure of POmAb, a Type-I anti-prothrombin antiphospho... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8uf7 | |||||||||
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Title | Cryo-EM structure of POmAb, a Type-I anti-prothrombin antiphospholipid antibody, bound to kringle-1 of human prothrombin | |||||||||
Components |
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Keywords | BLOOD CLOTTING / Autoimmunity / Thrombosis / Complex / Immunoglobulin / Coagulation factor / Anticoagulation / Inhibitor | |||||||||
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 platelet activation / negative regulation of astrocyte differentiation / 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 / fibrinolysis / regulation of cytosolic calcium ion concentration / 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 / blood coagulation / antimicrobial humoral immune response mediated by antimicrobial peptide / 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 | |||||||||
Biological species | Mus musculus (house mouse) Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Kumar, S. / Summers, B. / Basore, K. / Pozzi, N. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8uf7.cif.gz | 120.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8uf7.ent.gz | 85.4 KB | Display | PDB format |
PDBx/mmJSON format | 8uf7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uf/8uf7 ftp://data.pdbj.org/pub/pdb/validation_reports/uf/8uf7 | HTTPS FTP |
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-Related structure data
Related structure data | 42185MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Antibody | Mass: 23558.154 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster) |
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#2: Antibody | Mass: 23406.252 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster) |
#3: Protein | Mass: 65386.113 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P00734 |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of Fab fragment of POmAb with human prothrombin Type: COMPLEX Details: Fab fragment generated by proteolytic cleavage of POmAb IgG antibody in complex with open conformation of human prothrombin Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.12 MDa / Experimental value: YES |
Source (natural) | Organism: Mus musculus (house mouse) |
Source (recombinant) | Organism: Cricetulus griseus (Chinese hamster) |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 0.33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse particles. Complex purified by SEC. |
Specimen support | Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 84 K / Temperature (min): 82 K |
Image recording | Electron dose: 62.2 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) |
EM imaging optics | Spherical aberration corrector: Microscope is outfitted with a Cs image corrector with two hexapole elements. |
Image scans | Width: 4096 / Height: 4096 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176744 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||
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