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- PDB-8rew: CryoEM structure of human GARP-lTGFbeta1 in complex with a Fab fr... -

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Basic information

Entry
Database: PDB / ID: 8rew
TitleCryoEM structure of human GARP-lTGFbeta1 in complex with a Fab fragment derived from an activating antibody.
Components
  • (Transforming growth factor ...) x 2
  • (hFab LHT-22, ...) x 2
KeywordsIMMUNE SYSTEM / GARP / TGF-B1 / ACTIVATION / TREG / ANTIBODY
Function / homology
Function and homology information


establishment of protein localization to extracellular region / frontal suture morphogenesis / Influenza Virus Induced Apoptosis / adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains / positive regulation of microglia differentiation / regulation of interleukin-23 production / positive regulation of primary miRNA processing / morphogenesis of a branching structure / negative regulation of skeletal muscle tissue development / regulation of striated muscle tissue development ...establishment of protein localization to extracellular region / frontal suture morphogenesis / Influenza Virus Induced Apoptosis / adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains / positive regulation of microglia differentiation / regulation of interleukin-23 production / positive regulation of primary miRNA processing / morphogenesis of a branching structure / negative regulation of skeletal muscle tissue development / regulation of striated muscle tissue development / response to laminar fluid shear stress / embryonic liver development / heart valve morphogenesis / macrophage derived foam cell differentiation / TGFBR2 MSI Frameshift Mutants in Cancer / regulation of protein import into nucleus / regulation of blood vessel remodeling / transforming growth factor beta complex / cellular response to acetaldehyde / negative regulation of natural killer cell mediated cytotoxicity directed against tumor cell target / negative regulation of macrophage cytokine production / extracellular matrix assembly / negative regulation of hyaluronan biosynthetic process / connective tissue replacement involved in inflammatory response wound healing / type III transforming growth factor beta receptor binding / myofibroblast differentiation / TGFBR2 Kinase Domain Mutants in Cancer / positive regulation of exit from mitosis / positive regulation of isotype switching to IgA isotypes / secondary palate development / SMAD2/3 Phosphorylation Motif Mutants in Cancer / TGFBR1 KD Mutants in Cancer / odontoblast differentiation / positive regulation of mesenchymal stem cell proliferation / positive regulation of receptor signaling pathway via STAT / membrane protein intracellular domain proteolysis / positive regulation of extracellular matrix assembly / TGFBR3 regulates TGF-beta signaling / positive regulation of vasculature development / hyaluronan catabolic process / negative regulation of myoblast differentiation / ATP biosynthetic process / negative regulation of extracellular matrix disassembly / type II transforming growth factor beta receptor binding / cell-cell junction organization / positive regulation of branching involved in ureteric bud morphogenesis / positive regulation of cardiac muscle cell differentiation / response to salt / TGFBR1 LBD Mutants in Cancer / receptor catabolic process / regulatory T cell differentiation / negative regulation of cell-cell adhesion mediated by cadherin / negative regulation of biomineral tissue development / type I transforming growth factor beta receptor binding / receptor ligand inhibitor activity / positive regulation of chemotaxis / positive regulation of vascular permeability / positive regulation of mononuclear cell migration / oligodendrocyte development / negative regulation of interleukin-17 production / phosphate-containing compound metabolic process / face morphogenesis / sprouting angiogenesis / response to cholesterol / transforming growth factor beta binding / ureteric bud development / digestive tract development / positive regulation of chemokine (C-X-C motif) ligand 2 production / neural tube development / response to vitamin D / positive regulation of fibroblast migration / odontogenesis of dentin-containing tooth / aortic valve morphogenesis / RUNX3 regulates CDKN1A transcription / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of regulatory T cell differentiation / Molecules associated with elastic fibres / negative regulation of fat cell differentiation / negative regulation of neuroblast proliferation / Syndecan interactions / cellular response to insulin-like growth factor stimulus / ventricular cardiac muscle tissue morphogenesis / negative regulation of phagocytosis / positive regulation of interleukin-17 production / inner ear development / response to immobilization stress / negative regulation of cell cycle / chondrocyte differentiation / TGF-beta receptor signaling activates SMADs / positive regulation of protein metabolic process / positive regulation of collagen biosynthetic process / positive regulation of cell division / negative regulation of blood vessel endothelial cell migration / cellular response to dexamethasone stimulus / positive regulation of SMAD protein signal transduction / hematopoietic progenitor cell differentiation / epithelial to mesenchymal transition / vasculogenesis / positive regulation of endothelial cell apoptotic process / RUNX3 regulates p14-ARF
Similarity search - Function
Transforming growth factor beta-1 proprotein / Transforming growth factor-beta / Leucine rich repeat N-terminal domain / TGF-beta, propeptide / TGF-beta propeptide / Transforming growth factor beta, conserved site / TGF-beta family signature. / Transforming growth factor-beta-related / Transforming growth factor-beta (TGF-beta) family / Transforming growth factor-beta, C-terminal ...Transforming growth factor beta-1 proprotein / Transforming growth factor-beta / Leucine rich repeat N-terminal domain / TGF-beta, propeptide / TGF-beta propeptide / Transforming growth factor beta, conserved site / TGF-beta family signature. / Transforming growth factor-beta-related / Transforming growth factor-beta (TGF-beta) family / Transforming growth factor-beta, C-terminal / Transforming growth factor beta like domain / TGF-beta family profile. / Leucine-rich repeat N-terminal domain / Leucine rich repeat N-terminal domain / Leucine rich repeat, ribonuclease inhibitor type / Leucine-rich repeats, bacterial type / Cystine-knot cytokine / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Transforming growth factor beta-1 proprotein / Transforming growth factor beta activator LRRC32
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsFelix, J. / Lambert, F. / Marien, L. / van der Woning, B. / Savvides, S.N. / Lucas, S.
Funding support Belgium, 5items
OrganizationGrant numberCountry
Other privateFondation contre le Cancer / 2020-079
Other governmentARC / 19/24-098 Belgium
Fonds National de la Recherche Scientifique (FNRS)PDR / T.0145.21 Belgium
Fonds National de la Recherche Scientifique (FNRS)WELBIO / CR2019A-02R Belgium
Other privateSalus Sanguinis
CitationJournal: Cell Rep / Year: 2025
Title: Antibody-mediated TGF-β1 activation for the treatment of diseases caused by deleterious T cell activity.
Authors: Fanny Lambert / Jan Felix / Séverine Wautier / Emilie Dupré / Mathieu Jamez / Camille Michiels / Mélanie Gaignage / Lore Mariën / Manon Lesage / Bas van der Woning / Savvas N Savvides / Sophie Lucas /
Abstract: Transforming growth factor β1 (TGF-β1) is an immunosuppressive cytokine produced as a latent homodimer, in which mature TGF-β1 is encapsulated and kept inactive by the latency-associated peptide ...Transforming growth factor β1 (TGF-β1) is an immunosuppressive cytokine produced as a latent homodimer, in which mature TGF-β1 is encapsulated and kept inactive by the latency-associated peptide (LAP). The transmembrane protein GARP presents latent TGF-β1 on the surface of regulatory T cells (Tregs) to enable activation and release of mature TGF-β1 by integrins. Here, we derived monoclonal antibodies (mAbs) that activate latent TGF-β1 anchored on cells by a transmembrane protein. Biochemical and structural studies by electron cryo-microscopy (cryo-EM) reveal that such mAb-mediated activation requires bivalent binding close to the LAP dimerization interface and crosslinking of two membrane-bound GARP:TGF-β1 complexes on the same cell or across different cells. Administration of mAbs to mice with graft versus host disease reduced disease severity and increased survival. The therapeutic effect required Tregs. Collectively, our findings demonstrate that activation of membrane-bound TGF-β1 in vivo is achievable with mAbs, introducing new immunotherapeutic options for allo- or autoimmune diseases characterized by deleterious T cell activity insufficiently controlled by Tregs.
History
DepositionDec 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Mar 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0Mar 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transforming growth factor beta-1
B: Transforming growth factor beta-1
C: Transforming growth factor beta-1
D: Transforming growth factor beta-1
E: Transforming growth factor beta activator LRRC32
F: hFab LHT-22, Light Chain
G: hFab LHT-22, Heavy Chain
H: hFab LHT-22, Heavy Chain
I: hFab LHT-22, Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)356,56717
Polymers353,5399
Non-polymers3,0288
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area25270 Å2
ΔGint-67 kcal/mol
Surface area65600 Å2
MethodPISA

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Components

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Transforming growth factor ... , 2 types, 5 molecules ABCDE

#1: Protein
Transforming growth factor beta-1 / TGF-beta-1


Mass: 44383.051 Da / Num. of mol.: 4 / Fragment: LAP
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TGFB1, TGFB / Production host: Homo sapiens (human) / References: UniProt: P01137
#2: Protein Transforming growth factor beta activator LRRC32 / Garpin / Glycoprotein A repetitions predominant / GARP / Leucine-rich repeat-containing protein 32


Mass: 73261.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRC32, D11S833E / Production host: Homo sapiens (human) / References: UniProt: Q14392

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Antibody , 2 types, 4 molecules FIGH

#3: Antibody hFab LHT-22, Light Chain


Mass: 25104.023 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Homo sapiens (human)
#4: Antibody hFab LHT-22, Heavy Chain


Mass: 26268.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Homo sapiens (human)

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Sugars , 3 types, 8 molecules

#5: Polysaccharide alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c6-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: 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
#7: 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: 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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Details

Has ligand of interestN
Has protein modificationY

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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: human GAPR-lTGFbeta1 in complex with Fab LHT-22. / Type: COMPLEX / Entity ID: #2-#4 / Source: RECOMBINANT
Molecular weightValue: 260 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were acquired via PUXANO (https://puxano.com)
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 3.37 sec. / Electron dose: 61.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 13898
Details: A total of 6678 untilted movies were collected with a defocus of 0.8 to 2 micrometer. Following untilted data collection, 4020 movies were collected at 16 degree tilt and 3200 movies were ...Details: A total of 6678 untilted movies were collected with a defocus of 0.8 to 2 micrometer. Following untilted data collection, 4020 movies were collected at 16 degree tilt and 3200 movies were collected at 35 degree tilt, all using similar microscope settings.

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 320551 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6GFF

6gff


Accession code: 6GFF / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412682
ELECTRON MICROSCOPYf_angle_d0.73517270
ELECTRON MICROSCOPYf_dihedral_angle_d5.741840
ELECTRON MICROSCOPYf_chiral_restr0.0462032
ELECTRON MICROSCOPYf_plane_restr0.0072199

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