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- PDB-8bw0: Structure of CEACAM5 A3-B3 domain in Complex with Tusamitamab Fab -

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Basic information

Entry
Database: PDB / ID: 8bw0
TitleStructure of CEACAM5 A3-B3 domain in Complex with Tusamitamab Fab
Components
  • Carcinoembryonic antigen-related cell adhesion molecule 5
  • Tusamitamab Fab heavy Chain
  • Tusamitamab Light Chain
KeywordsCELL ADHESION / CEACAM5 / Tusamitamab / Cancer / cryo-EM / small molecular weight / Fab / A3-B3 / human membrane protein
Function / homology
Function and homology information


GPI anchor binding / homotypic cell-cell adhesion / negative regulation of myotube differentiation / Post-translational modification: synthesis of GPI-anchored proteins / heterophilic cell-cell adhesion / homophilic cell-cell adhesion / negative regulation of anoikis / side of membrane / Cell surface interactions at the vascular wall / basolateral plasma membrane ...GPI anchor binding / homotypic cell-cell adhesion / negative regulation of myotube differentiation / Post-translational modification: synthesis of GPI-anchored proteins / heterophilic cell-cell adhesion / homophilic cell-cell adhesion / negative regulation of anoikis / side of membrane / Cell surface interactions at the vascular wall / basolateral plasma membrane / apical plasma membrane / apoptotic process / negative regulation of apoptotic process / cell surface / protein homodimerization activity / extracellular exosome / extracellular region / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. ...: / Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Cell adhesion molecule CEACAM5
Similarity search - Component
Biological speciesMus sp. (mice)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsKumar, A. / Bertrand, T. / Rapisarda, C. / Rak, A.
Funding support France, 1items
OrganizationGrant numberCountry
Other private France
Citation
Journal: Nat Commun / Year: 2024
Title: Structural insights into epitope-paratope interactions of a monoclonal antibody targeting CEACAM5-expressing tumors.
Authors: Anand Kumar / Francis Duffieux / Marie Gagnaire / Chiara Rapisarda / Thomas Bertrand / Alexey Rak /
Abstract: Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are overexpressed in some tumor types. The antibody-drug conjugate tusamitamab ravtansine specifically recognizes the A3-B3 domains ...Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are overexpressed in some tumor types. The antibody-drug conjugate tusamitamab ravtansine specifically recognizes the A3-B3 domains of human CEACAM5 (hCEACAM5). To understand this specificity, here we map the epitope-paratope interface between the A3-B3 domains of hCEACAM5 (hCEACAM5) and the antigen-binding fragment of tusamitamab (tusa Fab). We use hydrogen/deuterium exchange mass spectrometry to identify the tusa Fab paratope, which involves heavy chain (HC) residues 101-109 and light chain residues 48-54 and 88-104. Using surface plasmon resonance, we demonstrate that alanine variants of HC residues 96-108 abolish binding to hCEACAM5, suggesting that these residues are critical for tusa-Fab-antigen complex formation. The cryogenic electron microscopy structure of the hCEACAM5- tusa Fab complex (3.11 Å overall resolution) reveals a discontinuous epitope involving residues in the A3-B3 domains and an N-linked mannose at residue Asn612. Conformational constraints on the epitope-paratope interface enable tusamitamab to target hCEACAM5 and distinguish CEACAM5 from other CEACAMs.
#1: Journal: Res Sq / Year: 2023
Title: Structural insights into epitope-paratope interactions of monoclonal antibody targeting CEACAM5-expressing tumors
Authors: Rak, A. / Kumar, A. / Duffi, F. / Gagnaire, M. / Rapisarda, C. / Bertrand, T.
History
DepositionDec 6, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 13, 2024Group: Data collection / Database references / Category: citation / citation_author / 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
H: Tusamitamab Fab heavy Chain
L: Tusamitamab Light Chain
C: Carcinoembryonic antigen-related cell adhesion molecule 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,7839
Polymers69,2143
Non-polymers2,5696
Water00
1


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

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Components

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Protein , 1 types, 1 molecules C

#3: Protein Carcinoembryonic antigen-related cell adhesion molecule 5 / Carcinoembryonic antigen / CEA / Meconium antigen 100


Mass: 20883.041 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A3-B3 domain / Source: (gene. exp.) Homo sapiens (human) / Gene: CEACAM5, CEA / Cell line (production host): HEK293FS / Production host: Homo sapiens (human) / References: UniProt: P06731

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Antibody , 2 types, 2 molecules HL

#1: Antibody Tusamitamab Fab heavy Chain


Mass: 24833.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Cell line (production host): HEK293FS / Production host: Homo sapiens (human)
#2: Antibody Tusamitamab Light Chain


Mass: 23497.072 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Cell line (production host): HEK293FS / Production host: Homo sapiens (human)

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Sugars , 3 types, 6 molecules

#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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: 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.: 1
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
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

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: hCEACAM5-A3B3-Tusamitamab Fab complex / Type: COMPLEX
Details: Human CEACAM5 a3-B3 domain in the comlex with the Tusamitamab Fab
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.07 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293FS
Buffer solutionpH: 7.4 / Details: Dulbeccos phosphate buffered saline
Buffer componentConc.: 1 X / Name: D-PBS
SpecimenConc.: 0.87 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0./1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: DARK FIELD / Nominal magnification: 240000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.72 sec. / Electron dose: 62 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5072

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.2particle selection
4cryoSPARC3.2CTF correction
7UCSF Chimera1.15model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2614488 / Details: Picked using blob picker
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213470 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033278
ELECTRON MICROSCOPYf_angle_d0.6324475
ELECTRON MICROSCOPYf_dihedral_angle_d11.99523
ELECTRON MICROSCOPYf_chiral_restr0.051539
ELECTRON MICROSCOPYf_plane_restr0.008560

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