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- PDB-8g6i: Coagulation factor VIII bound to a patient-derived anti-C1 domain... -

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Basic information

Entry
Database: PDB / ID: 8g6i
TitleCoagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
Components
  • Coagulation factor VIII chimera from human and pig
  • NB33 heavy chain
  • NB33 light chain
KeywordsBLOOD CLOTTING / Immune system
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 / COPII-coated ER to Golgi transport vesicle / Defective factor IX causes thrombophilia ...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 / COPII-coated ER to Golgi transport vesicle / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / 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 / endoplasmic reticulum lumen / copper ion binding / 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 / Cupredoxin / Galactose-binding-like domain superfamily
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: 4.23 Å
AuthorsChilders, K.C. / Davulcu, O. / Haynes, R.M. / Lollar, P. / Doering, C.B. / Coxon, C.H. / Spiegel, P.C.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R15HL135658 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)U54HL141981 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R44HL117511 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R44HL110448 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Blood / Year: 2023
Title: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor.
Authors: Kenneth C Childers / Nathan G Avery / Kevin A Estrada Alamo / Omar Davulcu / Rose Marie Haynes / Pete Lollar / Christopher B Doering / Carmen H Coxon / P Clint Spiegel /
Abstract: The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in ...The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1-mediated clearance.
History
DepositionFeb 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 30, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.initial_refinement_model_id / _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
A: Coagulation factor VIII chimera from human and pig
B: NB33 light chain
C: NB33 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,0545
Polymers214,2053
Non-polymers8492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Coagulation factor VIII chimera from human and pig / Procoagulant component


Mass: 168287.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This protein is a chimera of human+pig coagulation factor VIII (PDB: 6MF0).
Source: (gene. exp.) Sus scrofa (pig), (gene. exp.) Homo sapiens (human)
Gene: F8, CF8, F8, F8C / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P12263, UniProt: P00451
#2: Antibody NB33 light chain


Mass: 22972.436 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 NB33 heavy chain


Mass: 22945.660 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 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}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: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
Type: COMPLEX
Details: Coagulation factor VIII (molecule 1) is a chimera of human+pig proteins which was expressed in CHO cells.
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.4 / Details: 150 mM NaCl, 20 mM HEPES pH 7.4
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 / Details: This sample was monodisperse.
Specimen supportGrid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK I / 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: 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: 800 nm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 1.565 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6993

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
9cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8628762
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65133 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6MF0

6mf0


Accession code: 6MF0 / Source name: PDB / Type: experimental model

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