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- PDB-9lfl: Cryo-EM structure of linker-extended biparatopic antibody BA1-GP4... -

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Basic information

Entry
Database: PDB / ID: 9lfl
TitleCryo-EM structure of linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
Components
  • TR92 heavy chain
  • TR92 light chain
  • TR96 heavy chain
  • TR96 light chain
  • Tumor necrosis factor receptor superfamily member 1B
KeywordsIMMUNE SYSTEM / Antibody / 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 / 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 ...glial cell-neuron signaling / regulation of cytokine production involved in immune response / tumor necrosis factor receptor superfamily complex / pulmonary valve development / RNA destabilization / 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 / negative regulation of cardiac muscle hypertrophy / regulation of neuroinflammatory response / positive regulation of myelination / positive regulation of membrane protein ectodomain proteolysis / regulation of myelination / Interleukin-10 signaling / regulation of T cell proliferation / positive regulation of oligodendrocyte differentiation / specific granule membrane / extrinsic apoptotic signaling pathway / TNFR2 non-canonical NF-kB pathway / tumor necrosis factor-mediated signaling pathway / intrinsic apoptotic signaling pathway in response to DNA damage / cellular response to lipopolysaccharide / Interleukin-4 and Interleukin-13 signaling / inflammatory response / membrane raft / ubiquitin protein ligase binding / Neutrophil degranulation / 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
Similarity search - Domain/homology
Tumor necrosis factor receptor superfamily member 1B
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsOtsuki, T. / Matsumoto, S. / Fujita, J. / Miyata, T. / Namba, K. / Kanada, R. / Okuno, Y. / Kamada, H. / Ohno, H. / Akiba, H.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP22ak0101099 Japan
Japan Agency for Medical Research and Development (AMED)JP24ama121042 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121003 Japan
Japan Society for the Promotion of Science (JSPS)JP24K01270 Japan
CitationJournal: J Biol Chem / Year: 2025
Title: Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region.
Authors: Takuya Otsuki / Shigeyuki Matsumoto / Junso Fujita / Tomoko Miyata / Keiichi Namba / Ryo Kanada / Yasushi Okuno / Haruhiko Kamada / Hiroaki Ohno / Hiroki Akiba /
Abstract: Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or ...Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.
History
DepositionJan 8, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 13, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tumor necrosis factor receptor superfamily member 1B
B: TR92 heavy chain
C: TR92 light chain
D: TR96 heavy chain
E: TR96 light chain


Theoretical massNumber of molelcules
Total (without water)109,9175
Polymers109,9175
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Tumor necrosis factor receptor superfamily member 1B / 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


Mass: 16747.895 Da / Num. of mol.: 1 / Mutation: A490V
Source method: isolated from a genetically manipulated source
Details: 1-178, Tumor necrosis factor receptor 1B, Homo sapiens; 179-543, Maltose/maltodextrin-binding periplasmic protein - Escherichia coli K-12; 545-550, hexahistidine tag
Source: (gene. exp.) Homo sapiens (human) / Gene: TNFRSF1B, TNFBR, TNFR2 / Cell line (production host): ExpiCHO / Production host: Homo sapiens (human) / References: UniProt: P20333
#2: Antibody TR92 heavy chain


Mass: 23777.564 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: TR92 antibody heavy chain / Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody TR92 light chain


Mass: 23242.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody TR96 heavy chain


Mass: 24093.518 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#5: Antibody TR96 light chain


Mass: 22055.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
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: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 0.71 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 4.87 sec. / Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5508
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4098

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2SerialEM4.1image acquisition
4cryoSPARC4.2.1CTF correction
7UCSF Chimera8.6.10model fitting
9PHENIX1.20.1_4487:model refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.1classification
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2971529
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178242 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3ALQ

3alq


Accession code: 3ALQ / Source name: PDB / Type: experimental model

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