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- PDB-8ty1: Cryo-EM structure of coagulation factor VIII bound to NB2E9 -

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Basic information

Entry
Database: PDB / ID: 8ty1
TitleCryo-EM structure of coagulation factor VIII bound to NB2E9
Components
  • Coagulation factor VIII
  • NB2E9 heavy chain
  • NB2E9 light chain
KeywordsBLOOD CLOTTING/INHIBITOR / Coagulation / Inhibitor antibody / factor VIII / BLOOD CLOTTING-INHIBITOR complex
Function / homology
Function and homology information


Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Defective F8 sulfation at Y1699 / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant ...Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Defective F8 sulfation at Y1699 / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / COPII-coated ER to Golgi transport vesicle / Defective F8 cleavage by thrombin / Common Pathway of Fibrin Clot Formation / Intrinsic Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / acute-phase response / Golgi lumen / blood coagulation / Platelet degranulation / oxidoreductase activity / copper ion binding / endoplasmic reticulum lumen / extracellular space / extracellular region / plasma membrane
Similarity search - Function
Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Multicopper oxidase, C-terminal / Multicopper oxidase ...Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Multicopper oxidase, C-terminal / Multicopper oxidase / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Multicopper oxidase, type 1 / Multicopper oxidase / Multicopper oxidase, N-terminal / Multicopper oxidase / Galactose-binding domain-like / Cupredoxin / Galactose-binding-like domain superfamily / Jelly Rolls / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Coagulation factor VIII / Coagulation factor VIII
Similarity search - Component
Biological speciesSus scrofa (pig)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsChilders, K.C. / Spiegel, P.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL141981 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL135658 United States
CitationJournal: J Thromb Haemost / Year: 2024
Title: Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain.
Authors: Kenneth C Childers / Ben Cowper / Jordan D Vaughan / Juliet R McGill / Omar Davulcu / Pete Lollar / Christopher B Doering / Carmen H Coxon / Paul C Spiegel /
Abstract: BACKGROUND: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody ...BACKGROUND: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined.
OBJECTIVES: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition.
METHODS: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy.
RESULTS: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis ...RESULTS: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain.
CONCLUSION: These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.
History
DepositionAug 24, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.0Sep 13, 2023Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Coagulation factor VIII
B: NB2E9 light chain
C: NB2E9 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,4206
Polymers217,1733
Non-polymers2,2463
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: Protein Coagulation factor VIII / Procoagulant component


Mass: 168287.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig), (gene. exp.) Homo sapiens (human)
Gene: F8, F8, F8C, F8, CF8 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P12263, UniProt: P00451
#2: Antibody NB2E9 light chain


Mass: 23513.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody NB2E9 heavy chain


Mass: 25373.285 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#4: 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.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
Has ligand of interestN
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: Coagulation factor VIII bound to NB2E9 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.4 / Details: 20 mM HEPES (pH 7.4) and 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 400 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 2700 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.2.1CTF correction
8PHENIXmodel refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94935 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513870
ELECTRON MICROSCOPYf_angle_d0.82418818
ELECTRON MICROSCOPYf_dihedral_angle_d7.3461924
ELECTRON MICROSCOPYf_chiral_restr0.0482071
ELECTRON MICROSCOPYf_plane_restr0.0062380

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