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- PDB-8vc7: Crystal Structure of Human BTN2A1 ectodomain in complex with Anta... -

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Basic information

Entry
Database: PDB / ID: 8vc7
TitleCrystal Structure of Human BTN2A1 ectodomain in complex with Antagonist 2A1.9 Fab
Components
  • Butyrophilin subfamily 2 member A1
  • Human IgG1 Fragment Antibody Heavy Chain
  • Human IgG1 Fragment Antibody Light Chain
KeywordsSIGNALING PROTEIN / Inhibitor / Complex / Antibody / Immune Recognition
Function / homology
Function and homology information


Butyrophilin (BTN) family interactions / regulation of cytokine production / lipid metabolic process / T cell receptor signaling pathway / signaling receptor binding / external side of plasma membrane / plasma membrane
Similarity search - Function
Butyrophilin subfamily 1/2, SPRY/PRY domain / : / : / Butyrophilin subfamily 3 member A2-like, Ig-C domain / SPRY-associated domain / SPRY-associated / PRY / Butyrophylin-like, SPRY domain / SPRY domain / B30.2/SPRY domain ...Butyrophilin subfamily 1/2, SPRY/PRY domain / : / : / Butyrophilin subfamily 3 member A2-like, Ig-C domain / SPRY-associated domain / SPRY-associated / PRY / Butyrophylin-like, SPRY domain / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Concanavalin A-like lectin/glucanase domain superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Butyrophilin subfamily 2 member A1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.76 Å
AuthorsRamesh, A. / Roy, S. / Adams, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Sci Rep / Year: 2025
Title: Mapping the extracellular molecular architecture of the pAg-signaling complex with α-Butyrophilin antibodies.
Authors: Amrita Ramesh / Sobhan Roy / Tomasz Slezak / James Fuller / Hortencia Graves / Murad R Mamedov / Alexander Marson / Anthony A Kossiakoff / Erin J Adams /
Abstract: Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell ...Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex hampers Vγ9Vδ2 T cell immunotherapeutic efficacy. A panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified α-BTN3A1 mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation, and that antagonism may occur through occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, a panel of additional α-BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vγ9Vδ2 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist mAbs provide molecular insights into BTN2A1 epitopes critical for pAg-signaling.
History
DepositionDec 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Human IgG1 Fragment Antibody Light Chain
J: Human IgG1 Fragment Antibody Heavy Chain
C: Butyrophilin subfamily 2 member A1
A: Human IgG1 Fragment Antibody Light Chain
B: Human IgG1 Fragment Antibody Heavy Chain
E: Butyrophilin subfamily 2 member A1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,74811
Polymers147,6416
Non-polymers1,1065
Water00
1
D: Human IgG1 Fragment Antibody Light Chain
J: Human IgG1 Fragment Antibody Heavy Chain
C: Butyrophilin subfamily 2 member A1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,4846
Polymers73,8213
Non-polymers6643
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Human IgG1 Fragment Antibody Light Chain
B: Human IgG1 Fragment Antibody Heavy Chain
E: Butyrophilin subfamily 2 member A1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,2635
Polymers73,8213
Non-polymers4422
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)44.915, 200.976, 206.684
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Antibody Human IgG1 Fragment Antibody Light Chain


Mass: 23258.783 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Light chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: VSSAV) (CDR2: IYSASSLY) (CDR3: SSSSLIT). N-terminal (S) residue is a linker/restriction ...Details: Light chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: VSSAV) (CDR2: IYSASSLY) (CDR3: SSSSLIT). N-terminal (S) residue is a linker/restriction enzyme artifact, disordered and not modeled.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Antibody Human IgG1 Fragment Antibody Heavy Chain


Mass: 24836.652 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Heavy chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: NLYSSSI) (CDR2: YIYPSSGYTS) (CDR3: YYYTRGYPDGMDY). N-terminal (EISE) is a ...Details: Heavy chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: NLYSSSI) (CDR2: YIYPSSGYTS) (CDR3: YYYTRGYPDGMDY). N-terminal (EISE) is a linker/restriction enzyme artifact and first residue of Fab, and disordered and not modeled. Middle (SSKSTSG) and C-terminal (KSCDKTHT) residues were disordered and not modeled.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Protein Butyrophilin subfamily 2 member A1


Mass: 25725.307 Da / Num. of mol.: 2 / Mutation: C219S
Source method: isolated from a genetically manipulated source
Details: BTN2A1 ectodomain with C219S mutation, and N- and C-terminal linkers. N-terminal (ADL) and C-terminal (VSPSGSGLEVLFQ) residues are disordered and not modeled. 3 glycans are denoted as NAG.
Source: (gene. exp.) Homo sapiens (human) / Gene: BTN2A1, BT2.1, BTF1 / Plasmid: pAc-GP67a / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7KYR7
#4: 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.: 5
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
Has ligand of interestN
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: 3.22 Å3/Da / Density % sol: 61.75 % / Description: Overlapping large plates
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8.3
Details: Optimized well-G9 from Morpheus MIDAS MD1-76-HT Screen: - 0.1M Buffer System 3, pH 8.3 (Buffer System 3 1M Stock: 50.3% 1M Bicine, 49.7% 1M Tris) - 0.1M Carboxylic Acids Mix (Carboxylic ...Details: Optimized well-G9 from Morpheus MIDAS MD1-76-HT Screen: - 0.1M Buffer System 3, pH 8.3 (Buffer System 3 1M Stock: 50.3% 1M Bicine, 49.7% 1M Tris) - 0.1M Carboxylic Acids Mix (Carboxylic Acids Mix 1M Stock: 0.2M Sodium formate; 0.2M Ammonium acetate; 0.2M Sodium citrate tribasic dihydrate; 0.2M Potassium sodium tartrate tetrahydrate; 0.2M Sodium oxamate) - 20% Precipitant mix 1 (Precipitant mix 1 60% Stock: 40% v/v PEG500 MME; 20% w/v PEG20000)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL14-1 / Wavelength: 1.195 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 31, 2023
Details: Mirror: Flat bent collimating Rh coated mirror, toroidal focussing mirror
RadiationMonochromator: Si (111) double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.195 Å / Relative weight: 1
ReflectionResolution: 2.76→39.46 Å / Num. obs: 49431 / % possible obs: 99.8 % / Redundancy: 8.9 % / Rmerge(I) obs: 0.258 / Rpim(I) all: 0.131 / Rrim(I) all: 0.29 / Net I/σ(I): 7.2
Reflection shellResolution: 2.76→2.85 Å / Redundancy: 9.3 % / Rmerge(I) obs: 1.424 / Num. unique obs: 4503 / Rpim(I) all: 0.711 / Rrim(I) all: 1.595 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.21rc1_5015refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.76→39.46 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 27.33 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2607 1998 4.04 %
Rwork0.2177 --
obs0.2194 49406 99.8 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.76→39.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9727 0 70 0 9797
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008
X-RAY DIFFRACTIONf_angle_d1.13
X-RAY DIFFRACTIONf_dihedral_angle_d15.8983534
X-RAY DIFFRACTIONf_chiral_restr0.0681558
X-RAY DIFFRACTIONf_plane_restr0.0091752
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.76-2.830.38151360.33963375X-RAY DIFFRACTION100
2.83-2.910.38311460.31133274X-RAY DIFFRACTION100
2.91-2.990.36651420.28823400X-RAY DIFFRACTION100
2.99-3.090.37681290.28833287X-RAY DIFFRACTION100
3.09-3.20.30311410.2783377X-RAY DIFFRACTION100
3.2-3.330.32991470.28783298X-RAY DIFFRACTION100
3.33-3.480.23971420.22513374X-RAY DIFFRACTION99
3.48-3.660.28691430.21883339X-RAY DIFFRACTION100
3.66-3.890.25771420.20873365X-RAY DIFFRACTION100
3.89-4.190.22721370.19163401X-RAY DIFFRACTION100
4.19-4.610.20341470.15233391X-RAY DIFFRACTION100
4.61-5.280.17271470.15383404X-RAY DIFFRACTION99
5.28-6.640.22821450.19343503X-RAY DIFFRACTION100
6.64-39.460.24191540.20493620X-RAY DIFFRACTION99

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