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- PDB-6y92: Structure of full-length CD20 in complex with Ofatumumab Fab -

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Basic information

Entry
Database: PDB / ID: 6y92
TitleStructure of full-length CD20 in complex with Ofatumumab Fab
Components
  • B-lymphocyte antigen CD20
  • Ofatumumab Fab heavy chain
  • Ofatumumab Fab light 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 proliferation / plasma membrane raft / B cell activation / 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 proliferation / plasma membrane raft / B cell activation / immunoglobulin binding / humoral immune response / B cell differentiation / B cell receptor signaling pathway / response to bacterium / protein tetramerization / 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
Membrane-spanning 4-domains subfamily A / CD20-like family / CD20-like family
Similarity search - Domain/homology
CHOLESTEROL HEMISUCCINATE / B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.73 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

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Structure visualization

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Assembly

Deposited unit
A: B-lymphocyte antigen CD20
D: Ofatumumab Fab light chain
C: Ofatumumab Fab heavy chain
H: Ofatumumab Fab heavy chain
L: Ofatumumab Fab light chain
B: B-lymphocyte antigen CD20
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,37712
Polymers132,4576
Non-polymers2,9206
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy, Negative staining
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12300 Å2
ΔGint-86 kcal/mol
Surface area71160 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: 19914.656 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Full-length wild type human CD20 / Source: (gene. exp.) Homo sapiens (human) / Cell: B-lymphocyte / Gene: MS4A1, CD20 / Plasmid: pcDNA3.1+ / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / References: UniProt: P11836
#2: Antibody Ofatumumab Fab light chain


Mass: 23174.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: IgG1 expression vector / Cell line (production host): HEK-293F / Production host: Homo sapiens (human)
#3: Antibody Ofatumumab Fab heavy chain


Mass: 23138.934 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: IgG1 expression vector / Cell line (production host): HEK-293F / Production host: Homo sapiens (human)
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestN

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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 Ofatumumab FabCOMPLEX#1-#30MULTIPLE SOURCES
2Full-length human CD20COMPLEX#11RECOMBINANT
3Ofatumumab FabCOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11
22NO
33NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
12Homo sapiens (human)9606
23Homo sapiens (human)9606extra cellular
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 ChlorideNaCl1
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

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated defocus min: 800 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 40 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2540

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fittingRigid Body fit
8Coot0.8.9.2model fittingManual fitting and extension
10cryoSPARC2.14.2initial Euler assignment
11cryoSPARC2.14.2final Euler assignment
12cryoSPARC2.14.2classification
13cryoSPARC2.14.23D reconstruction
14PHENIX1.17.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 919699
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99146 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13GIZ

3giz

H11-216
23GIZ

3giz

L11-211
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 87.63 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039731
ELECTRON MICROSCOPYf_angle_d0.6813253
ELECTRON MICROSCOPYf_dihedral_angle_d19.5531736
ELECTRON MICROSCOPYf_chiral_restr0.0421512
ELECTRON MICROSCOPYf_plane_restr0.0041652

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