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- EMDB-16279: Structure of CEACAM5 A3-B3 domain in Complex with Tusamitamab Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-16279
TitleStructure of CEACAM5 A3-B3 domain in Complex with Tusamitamab Fab
Map dataCryo EM map for the complex
Sample
  • Complex: hCEACAM5-A3B3-Tusamitamab Fab complex
    • Protein or peptide: Tusamitamab Fab heavy Chain
    • Protein or peptide: Tusamitamab Light Chain
    • Protein or peptide: Carcinoembryonic antigen-related cell adhesion molecule 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsCEACAM5 / Tusamitamab / Cancer / cell adhesion / 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 via plasma membrane cell adhesion molecules / homophilic cell adhesion via plasma membrane adhesion molecules / 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 via plasma membrane cell adhesion molecules / homophilic cell adhesion via plasma membrane adhesion molecules / 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 speciesHomo sapiens (human) / Mus sp. (mice)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsKumar A / Bertrand T / Rapisarda C / Rak A
Funding support France, 1 items
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, 2022-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16279.png

Downloads & links

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Map

FileDownload / File: emd_16279.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo EM map for the complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.16 Å/pix.
x 256 pix.
= 296.96 Å		1.16 Å/pix.
x 256 pix.
= 296.96 Å		1.16 Å/pix.
x 256 pix.
= 296.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-34.901577000000003 - 77.238235000000003
Average (Standard dev.)0.000000000002839 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_16279_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_16279_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : hCEACAM5-A3B3-Tusamitamab Fab complex

EntireName: hCEACAM5-A3B3-Tusamitamab Fab complex
Components
  • Complex: hCEACAM5-A3B3-Tusamitamab Fab complex
    • Protein or peptide: Tusamitamab Fab heavy Chain
    • Protein or peptide: Tusamitamab Light Chain
    • Protein or peptide: Carcinoembryonic antigen-related cell adhesion molecule 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: hCEACAM5-A3B3-Tusamitamab Fab complex

SupramoleculeName: hCEACAM5-A3B3-Tusamitamab Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Human CEACAM5 a3-B3 domain in the comlex with the Tusamitamab Fab
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 70 KDa

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Macromolecule #1: Tusamitamab Fab heavy Chain

MacromoleculeName: Tusamitamab Fab heavy Chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 24.833652 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLQESGPG LVKPGGSLSL SCAASGFVFS SYDMSWVRQT PERGLEWVAY ISSGGGITYA PSTVKGRFTV SRDNAKNTLY LQMNSLTSE DTAVYYCAAH YFGSSGPFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
EVQLQESGPG LVKPGGSLSL SCAASGFVFS SYDMSWVRQT PERGLEWVAY ISSGGGITYA PSTVKGRFTV SRDNAKNTLY LQMNSLTSE DTAVYYCAAH YFGSSGPFAY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT HHHHHH

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Macromolecule #2: Tusamitamab Light Chain

MacromoleculeName: Tusamitamab Light Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 23.497072 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPAS LSASVGDRVT ITCRASENIF SYLAWYQQKP GKSPKLLVYN TRTLAEGVPS RFSGSGSGTD FSLTISSLQP EDFATYYCQ HHYGTPFTFG SGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPAS LSASVGDRVT ITCRASENIF SYLAWYQQKP GKSPKLLVYN TRTLAEGVPS RFSGSGSGTD FSLTISSLQP EDFATYYCQ HHYGTPFTFG SGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #3: Carcinoembryonic antigen-related cell adhesion molecule 5

MacromoleculeName: Carcinoembryonic antigen-related cell adhesion molecule 5
type: protein_or_peptide / ID: 3 / Details: A3-B3 domain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.883041 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ELPKPSISSN NSKPVEDKDA VAFTCEPEAQ NTTYLWWVNG QSLPVSPRLQ LSNGNRTLTL FNVTRNDARA YVCGIQNSVS ANRSDPVTL DVLYGPDTPI ISPPDSSYLS GANLNLSCHS ASNPSPQYSW RINGIPQQHT QVLFIAKITP NNNGTYACFV S NLATGRNN ...String:
ELPKPSISSN NSKPVEDKDA VAFTCEPEAQ NTTYLWWVNG QSLPVSPRLQ LSNGNRTLTL FNVTRNDARA YVCGIQNSVS ANRSDPVTL DVLYGPDTPI ISPPDSSYLS GANLNLSCHS ASNPSPQYSW RINGIPQQHT QVLFIAKITP NNNGTYACFV S NLATGRNN SIVKSITVSA SGTSPGLSAH HHHHH

UniProtKB: Cell adhesion molecule CEACAM5

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.87 mg/mL
BufferpH: 7.4 / Component - Concentration: 1.0 X / Component - Name: D-PBS / Details: Dulbeccos phosphate buffered saline
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 5072 / Average exposure time: 4.72 sec. / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: OTHER / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 240000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 2614488 / Details: Picked using blob picker
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 213470
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3.2)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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