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- PDB-6y9a: Structure of full-length CD20 in complex with Obinutuzumab Fab -

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Basic information

Entry
Database: PDB / ID: 6y9a
TitleStructure of full-length CD20 in complex with Obinutuzumab Fab
Components
  • B-lymphocyte antigen CD20
  • Obinutuzumab Fab Light chain
  • Obinutuzumab Fab heavy chain
KeywordsMEMBRANE PROTEIN / Cancer immunotherapy / Therapeutic antibody
Function / homology
Function and homology information


store-operated calcium entry / positive regulation of calcium ion import across plasma membrane / calcium ion import into cytosol / epidermal growth factor receptor binding / B cell activation / B cell proliferation / plasma membrane raft / immunoglobulin binding / humoral immune response / B cell differentiation ...store-operated calcium entry / positive regulation of calcium ion import across plasma membrane / calcium ion import into cytosol / epidermal growth factor receptor binding / B cell activation / B cell proliferation / plasma membrane raft / immunoglobulin binding / humoral immune response / B cell differentiation / response to bacterium / protein tetramerization / B cell receptor signaling pathway / MHC class II protein complex binding / cell surface receptor signaling pathway / external side of plasma membrane / cell surface / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / plasma membrane
Similarity search - Function
CD20-like family / Membrane-spanning 4-domains subfamily A / CD20-like family
Similarity search - Domain/homology
B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKumar, A. / Reyes, N.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council (ERC)309657 France
CitationJournal: Science / Year: 2020
Title: Binding mechanisms of therapeutic antibodies to human CD20.
Authors: Anand Kumar / Cyril Planchais / Rémi Fronzes / Hugo Mouquet / Nicolas Reyes /
Abstract: Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I ...Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo-electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20.
History
DepositionMar 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 26, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2020Group: Structure summary / Category: struct / Item: _struct.pdbx_descriptor
Revision 1.2Sep 16, 2020Group: Structure summary / Category: entity / Item: _entity.pdbx_description

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Structure visualization

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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: B-lymphocyte antigen CD20
B: B-lymphocyte antigen CD20
H: Obinutuzumab Fab heavy chain
L: Obinutuzumab Fab Light chain


Theoretical massNumber of molelcules
Total (without water)84,8434
Polymers84,8434
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy, Negative staining
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7590 Å2
ΔGint-59 kcal/mol
Surface area41360 Å2
MethodPISA

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Components

#1: Protein B-lymphocyte antigen CD20 / B-lymphocyte surface antigen B1 / Bp35 / Leukocyte surface antigen Leu-16 / Membrane-spanning 4- ...B-lymphocyte surface antigen B1 / Bp35 / Leukocyte surface antigen Leu-16 / Membrane-spanning 4-domains subfamily A member 1


Mass: 18735.373 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell: B-Lymphocyte / Gene: MS4A1, CD20 / Plasmid: pcDNA3.1 + / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P11836
#2: Antibody Obinutuzumab Fab heavy chain


Mass: 23407.260 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: IgG1 Expression vector / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#3: Antibody Obinutuzumab Fab Light chain


Mass: 23964.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: IgG1 Expression vector / Cell line (production host): HEK293F / Production host: Homo sapiens (human)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Full-length human antigen CD20 in complex with ObinutuzumabCOMPLEXall0MULTIPLE SOURCES
2Full-length human CD20COMPLEX#11RECOMBINANT
3Obinutuzumab FabCOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11
22NO
33NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
12Homo sapiens (human)9606HEK-293FpcDNA3.1
23Homo sapiens (human)9606HEK-293FIgG1 expression vector
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2100 mMSodium ChlorideNaClSodium chloride1
30.0084 PercentGDN101GDN1
40.00168 PercentCholesterol hemisuccinateCHS1
SpecimenConc.: 1.2 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: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Grids were blot for 3.5 Sec.

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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 FIELDBright-field microscopy / Calibrated defocus min: 800 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 42.84 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10545
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fittingRigid Body fit
8Coot0.8.9.2model fittingRigid Body fit
10cryoSPARC2.14.2initial Euler assignment
11cryoSPARC2.14.2final Euler assignment
12cryoSPARC2.14.2classification
13cryoSPARC2.14.23D reconstruction
14PHENIX1.17.1model refinement
CTF correctionDetails: Gctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1316678
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63547 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13PP4

3pp4

H12-220
23PP4

3pp4

L11-219
36Y90

6y90

145-213
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 150.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036085
ELECTRON MICROSCOPYf_angle_d0.7958257
ELECTRON MICROSCOPYf_dihedral_angle_d7.899824
ELECTRON MICROSCOPYf_chiral_restr0.05945
ELECTRON MICROSCOPYf_plane_restr0.0081030

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