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- PDB-8hlb: Cryo-EM structure of biparatopic antibody Bp109-92 in complex wit... -

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Basic information

Entry
Database: PDB / ID: 8hlb
TitleCryo-EM structure of biparatopic antibody Bp109-92 in complex with TNFR2
Components
  • TR109 heavy chain
  • TR109 light chain
  • TR92 heavy chain
  • TR92 light chain
  • Tumor necrosis factor receptor superfamily member 1B,Maltose/maltodextrin-binding periplasmic protein
KeywordsIMMUNE SYSTEM / TNFR2 / biparatopic antibody / antagonist
Function / homology
Function and homology information


glial cell-neuron signaling / regulation of cytokine production involved in immune response / tumor necrosis factor receptor superfamily complex / pulmonary valve development / RNA destabilization / varicosity / aortic valve development / tumor necrosis factor receptor activity / negative regulation of extracellular matrix constituent secretion / positive regulation of apoptotic process involved in morphogenesis ...glial cell-neuron signaling / regulation of cytokine production involved in immune response / tumor necrosis factor receptor superfamily complex / pulmonary valve development / RNA destabilization / varicosity / aortic valve development / tumor necrosis factor receptor activity / negative regulation of extracellular matrix constituent secretion / positive regulation of apoptotic process involved in morphogenesis / regulation of T cell cytokine production / negative regulation of neuroinflammatory response / TNFs bind their physiological receptors / tumor necrosis factor binding / positive regulation of myelination / negative regulation of cardiac muscle hypertrophy / regulation of neuroinflammatory response / positive regulation of membrane protein ectodomain proteolysis / regulation of myelination / detection of maltose stimulus / Interleukin-10 signaling / maltose transport complex / carbohydrate transport / regulation of T cell proliferation / positive regulation of oligodendrocyte differentiation / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / specific granule membrane / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / tumor necrosis factor-mediated signaling pathway / extrinsic apoptotic signaling pathway / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / TNFR2 non-canonical NF-kB pathway / cellular response to growth factor stimulus / intrinsic apoptotic signaling pathway in response to DNA damage / outer membrane-bounded periplasmic space / cellular response to lipopolysaccharide / Interleukin-4 and Interleukin-13 signaling / periplasmic space / immune response / inflammatory response / membrane raft / neuronal cell body / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / perinuclear region of cytoplasm / extracellular region / membrane / plasma membrane
Similarity search - Function
Tumour necrosis factor receptor 1B / Tumor necrosis factor receptor 1B, N-terminal / : / TNFR/NGFR family cysteine-rich region domain profile. / TNFR/NGFR cysteine-rich region / TNFR/NGFR family cysteine-rich region signature. / Tumor necrosis factor receptor / nerve growth factor receptor repeats. / TNFR/NGFR cysteine-rich region / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site ...Tumour necrosis factor receptor 1B / Tumor necrosis factor receptor 1B, N-terminal / : / TNFR/NGFR family cysteine-rich region domain profile. / TNFR/NGFR cysteine-rich region / TNFR/NGFR family cysteine-rich region signature. / Tumor necrosis factor receptor / nerve growth factor receptor repeats. / TNFR/NGFR cysteine-rich region / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
Maltose/maltodextrin-binding periplasmic protein / Tumor necrosis factor receptor superfamily member 1B
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsAkiba, H. / Fujita, J. / Ise, T. / Nishiyama, K. / Miyata, T. / Kato, T. / Namba, K. / Ohno, H. / Kamada, H. / Nagata, S. / Tsumoto, K.
Funding support Japan, 10items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP22ak0101099 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101117 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121003 Japan
Japan Agency for Medical Research and Development (AMED)JP17pc0101020 Japan
Japan Science and TechnologyJPMJOP1861 Japan
Japan Society for the Promotion of Science (JSPS)JP21K06453 Japan
Japan Society for the Promotion of Science (JSPS)JP20K22630
Kyoto University Foundation Japan
Takeda Science Foundation Japan
JEOL YOKOGUSHI Research Alliance Laboratories of Osaka University Japan
CitationJournal: Commun Biol / Year: 2023
Title: Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.
Authors: Hiroki Akiba / Junso Fujita / Tomoko Ise / Kentaro Nishiyama / Tomoko Miyata / Takayuki Kato / Keiichi Namba / Hiroaki Ohno / Haruhiko Kamada / Satoshi Nagata / Kouhei Tsumoto /
Abstract: Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different ...Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.
History
DepositionNov 29, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 4, 2023Provider: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tumor necrosis factor receptor superfamily member 1B,Maltose/maltodextrin-binding periplasmic protein
B: TR109 heavy chain
C: TR109 light chain
D: TR92 heavy chain
E: TR92 light chain


Theoretical massNumber of molelcules
Total (without water)158,5145
Polymers158,5145
Non-polymers00
Water00
1


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

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Components

#1: Protein Tumor necrosis factor receptor superfamily member 1B,Maltose/maltodextrin-binding periplasmic protein / Tumor necrosis factor receptor 2 / TNF-R2 / Tumor necrosis factor receptor type II / TNF-RII / TNFR- ...Tumor necrosis factor receptor 2 / TNF-R2 / Tumor necrosis factor receptor type II / TNF-RII / TNFR-II / p75 / p80 TNF-alpha receptor / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 60447.305 Da / Num. of mol.: 1 / Mutation: A490V
Source method: isolated from a genetically manipulated source
Details: fusion protein of TNFR2 from human fused with MalE from E.Coli K-12
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: TNFRSF1B, TNFBR, TNFR2, malE, b4034, JW3994 / Production host: Homo sapiens (human) / References: UniProt: P20333, UniProt: P0AEX9
#2: Antibody TR109 heavy chain


Mass: 25080.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody TR109 light chain


Mass: 23852.311 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Antibody TR92 heavy chain


Mass: 25543.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody TR92 light chain


Mass: 23590.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
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: Cryo-EM structure of biparatopic antibody Bp109-92 in complex with TNFR2
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: 1x PBS
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 4.9 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARC3.3.2CTF correction
7UCSF Chimera1.15model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2218071
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100391 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 3ALQ

3alq


Pdb chain-ID: A / Accession code: 3ALQ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 60.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00297639
ELECTRON MICROSCOPYf_angle_d0.671910398
ELECTRON MICROSCOPYf_chiral_restr0.04391172
ELECTRON MICROSCOPYf_plane_restr0.00541330
ELECTRON MICROSCOPYf_dihedral_angle_d13.63052742

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