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- PDB-8r8d: Cryo-EM structure of coagulation factor beta-XIIa in complex with... -

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Basic information

Entry
Database: PDB / ID: 8r8d
TitleCryo-EM structure of coagulation factor beta-XIIa in complex with the garadacimab Fab fragment (symmetric dimer)
Components
  • Coagulation factor XII
  • Garadacimab heavy chain variable region
  • Garadacimab light chain variable region
KeywordsBLOOD CLOTTING / Complex / coagulation / trypsin-like serine protease / hereditary angioedema (HAE)
Function / homology
Function and homology information


coagulation factor XIIa / plasma kallikrein-kinin cascade / Factor XII activation / Defective SERPING1 causes hereditary angioedema / response to misfolded protein / positive regulation of plasminogen activation / blood coagulation, intrinsic pathway / positive regulation of fibrinolysis / misfolded protein binding / zymogen activation ...coagulation factor XIIa / plasma kallikrein-kinin cascade / Factor XII activation / Defective SERPING1 causes hereditary angioedema / response to misfolded protein / positive regulation of plasminogen activation / blood coagulation, intrinsic pathway / positive regulation of fibrinolysis / misfolded protein binding / zymogen activation / Defective factor XII causes hereditary angioedema / protein autoprocessing / positive regulation of blood coagulation / rough endoplasmic reticulum / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / protein processing / blood coagulation / : / innate immune response / serine-type endopeptidase activity / calcium ion binding / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Coagulation factor XII/hepatocyte growth factor activator / Fibronectin type I domain / Fibronectin, type I / Fibronectin type-I domain signature. / Fibronectin type-I domain profile. / Fibronectin type 1 domain / Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. ...Coagulation factor XII/hepatocyte growth factor activator / Fibronectin type I domain / Fibronectin, type I / Fibronectin type-I domain signature. / Fibronectin type-I domain profile. / Fibronectin type 1 domain / 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 / EGF-like domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / : / Kringle-like fold / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Coagulation factor XII
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsDrulyte, I.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2024
Title: Structural basis for the inhibition of βFXIIa by garadacimab.
Authors: Ieva Drulyte / Rajesh Ghai / Saw Yen Ow / Eugene A Kapp / Adam J Quek / Con Panousis / Michael J Wilson / Andrew D Nash / Matthias Pelzing /
Abstract: Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of ...Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.
History
DepositionNov 29, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin ...citation / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Coagulation factor XII
E: Garadacimab heavy chain variable region
I: Garadacimab light chain variable region
H: Garadacimab heavy chain variable region
L: Garadacimab light chain variable region
A: Coagulation factor XII
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,9548
Polymers148,8406
Non-polymers2,1142
Water32418
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Coagulation factor XII


Mass: 26537.750 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F12 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: P00748
#2: Antibody Garadacimab heavy chain variable region


Mass: 25120.281 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293 / Production host: Homo sapiens (human)
#3: Antibody Garadacimab light chain variable region


Mass: 22762.174 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293 / Production host: Homo sapiens (human)
#4: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O
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: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.174 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: Expi293
Buffer solutionpH: 5.5 / Details: 10 mM Na Acetate, 100 mM NaCl pH 5.5
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Immediately before blotting and plunge freezing, fluorinated octyl maltoside (FOM) was added to the sample to the final concentration of 0.005%-0.01% (w/v)
Specimen supportDetails: plasma current 20 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / 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 magnification: 165000 X / Nominal defocus max: 1250 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4000 / Details: 2000 with 0.005% FOM and 2000 with 0.01% FOM
EM imaging opticsEnergyfilter name: TFS Selectris
Details: Electron source E-CFEG (cold-FEG), energy filter Selectris X
Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU3image acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 271636
Details: Particles were picked from each of the datasets independently using a blob picker using 50-170 A diameter. 124767 particles were picked from the 0.005% FOM dataset and 146869 particles from ...Details: Particles were picked from each of the datasets independently using a blob picker using 50-170 A diameter. 124767 particles were picked from the 0.005% FOM dataset and 146869 particles from the 0.01% FOM dataset.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135296 / Symmetry type: POINT

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