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- PDB-5j13: Structural basis for TSLP antagonism by the therapeutic antibody ... -

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Basic information

Entry
Database: PDB / ID: 5j13
TitleStructural basis for TSLP antagonism by the therapeutic antibody Tezepelumab (MEDI9929 / AMG-157)
Components
  • Thymic stromal lymphopoietin
  • anti-TSLP Fab-fragment, heavy chain
  • anti-TSLP Fab-fragment, light chain
KeywordsIMMUNE SYSTEM / cytokine inflammation TSLP antibody
Function / homology
Function and homology information


positive regulation of chemokine (C-C motif) ligand 1 production / interleukin-7 receptor binding / positive regulation of granulocyte colony-stimulating factor production / positive regulation of mast cell activation / positive regulation of cytokine-mediated signaling pathway / positive regulation of receptor signaling pathway via STAT / interleukin-7-mediated signaling pathway / positive regulation of interleukin-13 production / positive regulation of interleukin-5 production / positive regulation of interleukin-10 production ...positive regulation of chemokine (C-C motif) ligand 1 production / interleukin-7 receptor binding / positive regulation of granulocyte colony-stimulating factor production / positive regulation of mast cell activation / positive regulation of cytokine-mediated signaling pathway / positive regulation of receptor signaling pathway via STAT / interleukin-7-mediated signaling pathway / positive regulation of interleukin-13 production / positive regulation of interleukin-5 production / positive regulation of interleukin-10 production / cell surface receptor signaling pathway via JAK-STAT / defense response to fungus / positive regulation of chemokine production / Interleukin-7 signaling / cytokine activity / positive regulation of interleukin-6 production / cytokine-mediated signaling pathway / positive regulation of inflammatory response / antimicrobial humoral immune response mediated by antimicrobial peptide / defense response to Gram-negative bacterium / positive regulation of cell population proliferation / negative regulation of apoptotic process / extracellular space / extracellular region
Similarity search - Function
Thymic stromal lymphopoietin / Thymic stromal lymphopoietin / Thymic stromal lymphopoietin superfamily / Thymic stromal lymphopoietin / Growth Hormone; Chain: A; / Immunoglobulins / Up-down Bundle / Immunoglobulin-like / Sandwich / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Thymic stromal lymphopoietin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.298 Å
AuthorsVerstraete, K. / Savvides, S.N.
CitationJournal: Nat Commun / Year: 2017
Title: Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.
Authors: Kenneth Verstraete / Frank Peelman / Harald Braun / Juan Lopez / Dries Van Rompaey / Ann Dansercoer / Isabel Vandenberghe / Kris Pauwels / Jan Tavernier / Bart N Lambrecht / Hamida Hammad / ...Authors: Kenneth Verstraete / Frank Peelman / Harald Braun / Juan Lopez / Dries Van Rompaey / Ann Dansercoer / Isabel Vandenberghe / Kris Pauwels / Jan Tavernier / Bart N Lambrecht / Hamida Hammad / Hans De Winter / Rudi Beyaert / Guy Lippens / Savvas N Savvides /
Abstract: The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma ...The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.
History
DepositionMar 28, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Apr 5, 2017Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2017Group: Database references
Revision 1.2Feb 28, 2018Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.gene_src_common_name / _entity_src_gen.host_org_common_name ..._entity_src_gen.gene_src_common_name / _entity_src_gen.host_org_common_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain / _entity_src_gen.pdbx_host_org_variant
Revision 1.3Jan 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thymic stromal lymphopoietin
B: anti-TSLP Fab-fragment, light chain
C: anti-TSLP Fab-fragment, heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,1216
Polymers70,8333
Non-polymers2883
Water1,946108
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5260 Å2
ΔGint-68 kcal/mol
Surface area23950 Å2
MethodPISA
Unit cell
Length a, b, c (Å)51.726, 51.726, 370.028
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number153
Space group name H-MP3212
Components on special symmetry positions
IDModelComponents
11B-302-

SO4

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Components

#1: Protein Thymic stromal lymphopoietin


Mass: 16523.906 Da / Num. of mol.: 1
Mutation: Residues 127 to 131 were deleted in the construct used for crystallisation.
Source method: isolated from a genetically manipulated source
Details: Before crystallisation, the N-terminal His-tag (residues 1 - 17, MGSSHHHHHHSSGLVPR) was removed by thrombin cleavage. Residues 127 to 131 of TSLP (127-RRKRK-131) (according to the reference ...Details: Before crystallisation, the N-terminal His-tag (residues 1 - 17, MGSSHHHHHHSSGLVPR) was removed by thrombin cleavage. Residues 127 to 131 of TSLP (127-RRKRK-131) (according to the reference sequence numbering scheme for TSLP) were deleted in the construct used for crystallization.
Source: (gene. exp.) Homo sapiens (human) / Gene: TSLP / Plasmid: pET15b-hTSLPdelta127-131
Details (production host): ORF cloned between NdeI and BamHI sites
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q969D9
#2: Antibody anti-TSLP Fab-fragment, light chain


Mass: 25870.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The signal peptide (residues 1 - 28) is removed from the mature protein.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pHLsec
Details (production host): ORF cloned between AgeI and KpnI sites
Cell line (production host): HEK-293T / Production host: Homo sapiens (human)
#3: Antibody anti-TSLP Fab-fragment, heavy chain


Mass: 28438.221 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The signal peptide (residues 1 - 28) is removed from the mature protein.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pHLsec
Details (production host): ORF cloned between AgeI and KpnI sites
Cell line (production host): HEK-293T / Production host: Homo sapiens (human)
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 108 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.26 Å3/Da / Density % sol: 45.49 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 0.2 M ammonium sulfate 0.1 M sodium acetate pH 4.6 25% w/v polyethylene glycol 4000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.9801 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Dec 17, 2015
RadiationMonochromator: Si[111] / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9801 Å / Relative weight: 1
ReflectionResolution: 2.298→55 Å / Num. obs: 25284 / % possible obs: 97 % / Redundancy: 8.4 % / Biso Wilson estimate: 47.33 Å2 / CC1/2: 0.998 / Rrim(I) all: 0.11 / Net I/σ(I): 14.23
Reflection shellResolution: 2.298→2.44 Å / Redundancy: 4.2 % / Mean I/σ(I) obs: 1.6 / Rrim(I) all: 0.747 / % possible all: 83.8

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
XDSVERSION Oct 15, 2015data reduction
PHASER2.5.7phasing
Coot0.8.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4HK0, chain B; 4HIE, chain B

4hk0

4hie


Resolution: 2.298→44.796 Å / SU ML: 0.3 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 23.26
Details: - rigid body refinement (single rigid body per domain) - Iterative cycles of refinement, model (re)building and validation xyz coordinate refinement real-space refinement individual B- ...Details: - rigid body refinement (single rigid body per domain) - Iterative cycles of refinement, model (re)building and validation xyz coordinate refinement real-space refinement individual B-factors riding hydrogens optimisation of X-ray/stereochemistry weight optimisation of X-ray/ADP weight structure validation in COOT, Phenix (Molprobity) and PDB_REDO
RfactorNum. reflection% reflection
Rfree0.215 1265 5 %
Rwork0.1891 --
obs0.1904 25275 96.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.298→44.796 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3987 0 15 108 4110
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034116
X-RAY DIFFRACTIONf_angle_d0.7655607
X-RAY DIFFRACTIONf_dihedral_angle_d12.3871425
X-RAY DIFFRACTIONf_chiral_restr0.028635
X-RAY DIFFRACTIONf_plane_restr0.004712
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2978-2.38970.39141160.30782190X-RAY DIFFRACTION81
2.3897-2.49850.29351310.282498X-RAY DIFFRACTION92
2.4985-2.63020.28411440.26382727X-RAY DIFFRACTION100
2.6302-2.7950.27031420.23412696X-RAY DIFFRACTION100
2.795-3.01070.23691440.23092734X-RAY DIFFRACTION100
3.0107-3.31360.2191440.21312740X-RAY DIFFRACTION100
3.3136-3.79290.20451440.182739X-RAY DIFFRACTION100
3.7929-4.77780.18521470.14452794X-RAY DIFFRACTION100
4.7778-44.80450.18731530.16092892X-RAY DIFFRACTION100

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