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- PDB-7sht: Structure of a partially disrupted IgE high affinity receptor com... -

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Basic information

Entry
Database: PDB / ID: 7sht
TitleStructure of a partially disrupted IgE high affinity receptor complex bound to an omalizumab variant
Components
  • (clone_7 Variable fragment ...) x 2
  • High affinity immunoglobulin epsilon receptor subunit alpha
  • Immunoglobulin heavy constant epsilon
KeywordsIMMUNE SYSTEM / IgE / allergy / Xolair / Omalizumab
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.29 Å
AuthorsPennington, L.F. / Jardetzky, T.S.
Funding support United States, 2items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 15, 2021Provider: repository / Type: Initial release

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Structure visualization

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  • EMDB-25136
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Assembly

Deposited unit
A: High affinity immunoglobulin epsilon receptor subunit alpha
B: Immunoglobulin heavy constant epsilon
D: Immunoglobulin heavy constant epsilon
H: clone_7 Variable fragment heavy chain
J: clone_7 Variable fragment heavy chain
K: clone_7 Variable fragment light chain
L: clone_7 Variable fragment light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,77214
Polymers150,2757
Non-polymers3,4967
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules ABD

#1: Protein High affinity immunoglobulin epsilon receptor subunit alpha / Fc-epsilon RI-alpha / FcERI / IgE Fc receptor subunit alpha


Mass: 22541.799 Da / Num. of mol.: 1 / Fragment: extracellular portion / Mutation: W156C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FCER1A, FCE1A / Production host: Homo sapiens (human) / References: UniProt: P12319
#2: Protein Immunoglobulin heavy constant epsilon / Ig epsilon chain C region / Ig epsilon chain C region ND


Mass: 35773.160 Da / Num. of mol.: 2 / Mutation: G335C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGHE / Production host: Homo sapiens (human) / References: UniProt: P01854

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Antibody , 2 types, 4 molecules HJKL

#3: Antibody clone_7 Variable fragment heavy chain


Mass: 13451.814 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Antibody clone_7 Variable fragment light chain


Mass: 14641.863 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Sugars , 4 types, 7 molecules

#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2-2-2/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

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

ComponentName: Locked complex of IgE-Fc (G335C) and alpha chain of the high affinity IgE receptor (W156C) bound to clone_7 scFV
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 2 sec. / Electron dose: 44 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 523

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Processing

Software
NameVersionClassificationNB
PHENIX(1.14_3260:phenix.real_space_refine)refinement
PDB_EXTRACT3.25data extraction
EM software
IDNameCategory
4cryoSPARCCTF correction
7PHENIXmodel fitting
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24396 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Initial 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 ...Details: Initial 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.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
11F6A

1f6a

A11-329
21F6A

1f6a

B1335-544
34J4P

4j4p

B11-329
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 756.63 Å2 / Biso mean: 344.0879 Å2 / Biso min: 20 Å2

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