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- EMDB-25136: Structure of a partially disrupted IgE high affinity receptor com... -

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Basic information

Entry
Database: EMDB / ID: EMD-25136
TitleStructure of a partially disrupted IgE high affinity receptor complex bound to an omalizumab variant
Map data
Sample
  • Complex: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV
    • Protein or peptide: High affinity immunoglobulin epsilon receptor subunit alpha
    • Protein or peptide: Immunoglobulin heavy constant epsilon
    • Protein or peptide: clone_7 Variable fragment heavy chain
    • Protein or peptide: clone_7 Variable fragment light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


high-affinity IgE receptor activity / IgE B cell receptor complex / adaptive immune memory response / primary adaptive immune response / type I hypersensitivity / B cell antigen processing and presentation / Fc receptor-mediated immune complex endocytosis / eosinophil degranulation / IgE immunoglobulin complex / macrophage activation ...high-affinity IgE receptor activity / IgE B cell receptor complex / adaptive immune memory response / primary adaptive immune response / type I hypersensitivity / B cell antigen processing and presentation / Fc receptor-mediated immune complex endocytosis / eosinophil degranulation / IgE immunoglobulin complex / macrophage activation / IgE binding / type 2 immune response / Fc epsilon receptor (FCERI) signaling / antibody-dependent cellular cytotoxicity / mast cell degranulation / B cell proliferation / macrophage differentiation / immunoglobulin mediated immune response / Role of LAT2/NTAL/LAB on calcium mobilization / immunoglobulin complex, circulating / immunoglobulin receptor binding / FCERI mediated Ca+2 mobilization / complement activation, classical pathway / FCERI mediated MAPK activation / antigen binding / B cell receptor signaling pathway / FCERI mediated NF-kB activation / antibacterial humoral response / Interleukin-4 and Interleukin-13 signaling / adaptive immune response / cell surface receptor signaling pathway / blood microparticle / inflammatory response / immune response / external side of plasma membrane / cell surface / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin subtype / Immunoglobulin / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set ...Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin subtype / Immunoglobulin / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Immunoglobulin heavy constant epsilon / High affinity immunoglobulin epsilon receptor subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.29 Å
AuthorsPennington LF / Jardetzky TS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI115469 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5F30AI120510-03 United States
CitationJournal: Nat Commun / Year: 2021
Title: Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex.
Authors: Luke F Pennington / Pascal Gasser / Silke Kleinboelting / Chensong Zhang / Georgios Skiniotis / Alexander Eggel / Theodore S Jardetzky /
Abstract: Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional ...Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.
History
DepositionOct 11, 2021-
Header (metadata) releaseDec 15, 2021-
Map releaseDec 15, 2021-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sht
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25136.png

Downloads & links

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Map

FileDownload / File: emd_25136.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8521 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.467892 - 1.032226
Average (Standard dev.)0.0063388236 (±0.060340285)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 218.1376 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.85210156250.85210156250.8521015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z218.138218.138218.138
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.4681.0320.006

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Supplemental data

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Sample components

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Entire : Locked complex of IgE-Fc (G335C) and alpha chain of the high affi...

EntireName: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV
Components
  • Complex: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV
    • Protein or peptide: High affinity immunoglobulin epsilon receptor subunit alpha
    • Protein or peptide: Immunoglobulin heavy constant epsilon
    • Protein or peptide: clone_7 Variable fragment heavy chain
    • Protein or peptide: clone_7 Variable fragment light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Locked complex of IgE-Fc (G335C) and alpha chain of the high affi...

SupramoleculeName: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: High affinity immunoglobulin epsilon receptor subunit alpha

MacromoleculeName: High affinity immunoglobulin epsilon receptor subunit alpha
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.541799 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: APMAEGGGQN HHHHHHHHGG ENLYFQGGSP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSG EYKCQHQQVN ESEPVYLEVF SDWLLLQASA EVVMEGQPLF LRCHGWRNWD VYKVIYYKDG EALKYWYENH N ISITNATV ...String:
APMAEGGGQN HHHHHHHHGG ENLYFQGGSP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSG EYKCQHQQVN ESEPVYLEVF SDWLLLQASA EVVMEGQPLF LRCHGWRNWD VYKVIYYKDG EALKYWYENH N ISITNATV EDSGTYYCTG KVCQLDYESE PLNITVIKA

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Macromolecule #2: Immunoglobulin heavy constant epsilon

MacromoleculeName: Immunoglobulin heavy constant epsilon / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.77316 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ASFTPPTVKI LQSSCDGGGH FPPTIQLLCL VSGYTPGTIN ITWLEDGQVM DVDLSTASTT QEGELASTQS ELTLSQKHWL SDRTYTCQV TYQGHTFEDS TKKCADSNPR CVSAYLSRPS PFDLFIRKSP TITCLVVDLA PSKGTVNLTW SRASGKPVNH S TRKEEKQR ...String:
ASFTPPTVKI LQSSCDGGGH FPPTIQLLCL VSGYTPGTIN ITWLEDGQVM DVDLSTASTT QEGELASTQS ELTLSQKHWL SDRTYTCQV TYQGHTFEDS TKKCADSNPR CVSAYLSRPS PFDLFIRKSP TITCLVVDLA PSKGTVNLTW SRASGKPVNH S TRKEEKQR NGTLTVTSTL PVGTRDWIEG ETYQCRVTHP HLPRALMRST TKTSGPRAAP EVYAFATPEW PGSRDKRTLA CL IQNFMPE DISVQWLHNE VQLPDARHST TQPRKTKGSG FFVFSRLEVT RAEWEQKDEF ICRAVHEAAS PSQTVQRAVS VNP GK

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Macromolecule #3: clone_7 Variable fragment heavy chain

MacromoleculeName: clone_7 Variable fragment heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.451814 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ASEVQLVESG GGLVQPDGSL RLSCAVSGYN ITSGYSWNWI RQTPGKGLEW VASVTYDGST NYNPSVKGRI TISRDGSKNT FYLQMNSLR AEDTAVYYCA KGNNYFGHWH FAVWGQGTLV TVSS

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Macromolecule #4: clone_7 Variable fragment light chain

MacromoleculeName: clone_7 Variable fragment light chain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.641863 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GSDIQLTQSP SSLSASVGDR VTITCRASKS VDSDGDSYMN WYQQKPGRAP KLLIYAASYL ESGVPSRFSG SGSGTHFTLT ISSLQPEDF ATYYCQQSHE DPYTFGQGTK VEIKGGSENL YFQGGSGHHH HHHHH

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 523 / Average exposure time: 2.0 sec. / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC
Startup modelType of model: OTHER
Details: Ab initio from preliminary relion models used at template for final refinements in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 24396
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
PDB IDChain

1f6a

chain_id: A, residue_range: 1-329

1f6a

chain_id: B, residue_range: 335-544

4j4p

chain_id: B, residue_range: 1-329
DetailsInitial local fitting done in Chimera, followed by auto dock, and a single round of real space refinement. Carbohydrates were automatically built in phenix when possible, and a handful of flagged outliers in geometry were fixed manually in Phenix.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7sht:
Structure of a partially disrupted IgE high affinity receptor complex bound to an omalizumab variant

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