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- PDB-7n0u: Complex of recombinant Bet v 1 with Fab fragment of REGN5713 -

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Basic information

Entry
Database: PDB / ID: 7n0u
TitleComplex of recombinant Bet v 1 with Fab fragment of REGN5713
Components
  • (REGN5713 Fab fragment ...) x 2
  • Recombinant Bet v 1
KeywordsALLERGEN / Birch pollen / allergy / neutralizing antibody / immunotherapy
Function / homology
Function and homology information


response to biotic stimulus / abscisic acid binding / abscisic acid-activated signaling pathway / protein phosphatase inhibitor activity / defense response / signaling receptor activity / nucleus / cytoplasm
Similarity search - Function
Pathogenesis-related protein Bet v I family / Pathogenesis-related proteins Bet v I family signature. / Bet v I type allergen / Bet v I/Major latex protein / Pathogenesis-related protein Bet v 1 family / START-like domain superfamily
Similarity search - Domain/homology
Major pollen allergen Bet v 1-A
Similarity search - Component
Biological speciesMus musculus (house mouse)
Betula pendula (European white birch)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsFranklin, M.C. / Romero Hernandez, A.
CitationJournal: J Allergy Clin Immunol / Year: 2022
Title: Targeting immunodominant Bet v 1 epitopes with monoclonal antibodies prevents the birch allergic response.
Authors: Amanda Atanasio / Matthew C Franklin / Vishal Kamat / Annabel Romero Hernandez / Ashok Badithe / Li-Hong Ben / Jennifer Jones / Joannie Bautista / George D Yancopoulos / William Olson / ...Authors: Amanda Atanasio / Matthew C Franklin / Vishal Kamat / Annabel Romero Hernandez / Ashok Badithe / Li-Hong Ben / Jennifer Jones / Joannie Bautista / George D Yancopoulos / William Olson / Andrew J Murphy / Matthew A Sleeman / Jamie M Orengo
Abstract: BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response.
OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein ...OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response.
METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy ...METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model.
RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- ...RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1.
CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous ...CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.
History
DepositionMay 26, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 19, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: REGN5713 Fab fragment heavy chain
L: REGN5713 Fab fragment light chain
C: Recombinant Bet v 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,1555
Polymers67,8383
Non-polymers3172
Water19811
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5690 Å2
ΔGint-46 kcal/mol
Surface area25760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)140.766, 140.766, 94.294
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

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Antibody , 2 types, 2 molecules HL

#1: Antibody REGN5713 Fab fragment heavy chain


Mass: 23560.197 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: VI3 humanized / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody REGN5713 Fab fragment light chain


Mass: 23534.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: VI3 humanized / Production host: Cricetulus griseus (Chinese hamster)

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Protein / Sugars , 2 types, 2 molecules C

#3: Protein Recombinant Bet v 1


Mass: 20743.148 Da / Num. of mol.: 1 / Mutation: F63L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Betula pendula (European white birch) / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P15494
#5: 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.: 1 / Source method: obtained synthetically / 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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Non-polymers , 2 types, 12 molecules

#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 11 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 3.98 Å3/Da / Density % sol: 69.06 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / Details: ammonium sulfate, sodium acetate, glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.987 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 28, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 2.99→100 Å / Num. obs: 22121 / % possible obs: 100 % / Redundancy: 9.3 % / Biso Wilson estimate: 64.48 Å2 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.039 / Rrim(I) all: 0.118 / Net I/σ(I): 17.7
Reflection shellResolution: 3→3.05 Å / Redundancy: 9 % / Rmerge(I) obs: 1.07 / Num. unique obs: 1100 / CC1/2: 0.72 / Rpim(I) all: 0.373 / Rrim(I) all: 1.13 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4A8U and in-house Fab structure

4a8u


Resolution: 3→46.08 Å / SU ML: 0.3137 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.2687
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.28 1039 4.75 %
Rwork0.2403 20833 -
obs0.2422 21872 98.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 69.93 Å2
Refinement stepCycle: LAST / Resolution: 3→46.08 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4437 0 19 11 4467
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00274558
X-RAY DIFFRACTIONf_angle_d0.64466202
X-RAY DIFFRACTIONf_chiral_restr0.0454703
X-RAY DIFFRACTIONf_plane_restr0.005794
X-RAY DIFFRACTIONf_dihedral_angle_d6.4084627
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.150.32881490.31532862X-RAY DIFFRACTION95.8
3.15-3.340.35131250.30632987X-RAY DIFFRACTION100
3.34-3.60.44081420.31722980X-RAY DIFFRACTION99.68
3.6-3.960.46191380.32922860X-RAY DIFFRACTION95.36
3.96-4.540.21631850.20152971X-RAY DIFFRACTION100
4.54-5.710.18011520.18163023X-RAY DIFFRACTION100
5.72-46.080.22331480.19373150X-RAY DIFFRACTION99.94
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.36665297815-0.129322987332-0.1984134468094.0716717931-1.412834031014.46469071780.128606657465-0.2966105287240.2868431003590.431105368662-0.08146040697560.00146747312098-0.2096912226370.217195000321-0.004961939396130.179429177923-0.09251533408540.00341441154890.353405939747-0.05096874702640.377445033341-62.248438579370.6483375412-17.2629108854
20.85084376408-0.209280060958-1.060395135635.081446881480.7244725264293.309968196470.161679155556-0.3789148404240.08285406344550.3917854427760.0238409802017-0.7517495690110.4527497655590.490422679551-0.1884425012180.4018315761970.0119251309935-0.1241061472870.722583431453-0.03341135796340.3455399146-48.495761946345.5163014461-8.67074003427
32.71098112349-0.0447728260327-0.1623899929294.92033548404-0.4866743733542.677191981440.2335679474330.09755808115020.14208793142-0.682231888495-0.133721010180.712566661580.504564392986-0.110913406427-0.05105350236790.425460013673-0.102801336689-0.1295037889620.395869219493-0.004607678710540.382512293603-70.082642774256.8835696995-32.9617512714
42.078061962191.381405153290.1066608285992.541021214911.164220862272.578669481230.229497469042-0.46242814243-0.2614163273881.13834564399-0.184672997247-0.2155666527671.154471303730.3480671825990.01970833663651.2948768190.105743881507-0.1438400701280.6817946012320.04265729481930.347149190654-56.52053973431.0342622523-8.86014483278
53.421289143770.1973784996451.306004813683.90577943184-0.323318780933.9178937322-0.1683319137110.06705312193960.832492550995-0.197530727815-0.05226856563150.869807736342-0.54238424406-0.569333181335-0.15569519390.3491448055550.0535386529556-0.09834860795870.4061292778-0.007913839462480.8490632416-72.067835898790.7691236905-36.1919109372
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11(chain H and resseq 2:115)HA2 - 1151 - 114
22(chain H and resseq 116:214)HA116 - 214115 - 207
33(chain L and resseq 1:107)LB1 - 1071 - 107
44(chain L and resseq 108:214)LB108 - 214108 - 214
55(chain C and resseq 1:159)CC1 - 1591 - 159

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