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

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Basic information

Entry
Database: PDB / ID: 8rex
TitleCryoEM structure of mouse GARP-lTGFbeta1 in complex with a Fab fragment derived from an activating antibody.
Components
  • (Transforming growth factor ...) x 2
  • (mFab LMT-12, ...) x 2
KeywordsIMMUNE SYSTEM / GARP / TGF-B1 / ACTIVATION / TREG / ANTIBODY
Function / homology
Function and homology information


regulation of CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation / Syndecan interactions / TGFBR3 regulates TGF-beta signaling / morphogenesis of a branching structure / RUNX3 regulates CDKN1A transcription / RUNX3 regulates p14-ARF / Molecules associated with elastic fibres / establishment of protein localization to extracellular region / CD4-positive, CD25-positive, alpha-beta regulatory T cell lineage commitment / connective tissue development ...regulation of CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation / Syndecan interactions / TGFBR3 regulates TGF-beta signaling / morphogenesis of a branching structure / RUNX3 regulates CDKN1A transcription / RUNX3 regulates p14-ARF / Molecules associated with elastic fibres / establishment of protein localization to extracellular region / CD4-positive, CD25-positive, alpha-beta regulatory T cell lineage commitment / connective tissue development / TGF-beta receptor signaling activates SMADs / frontal suture morphogenesis / Downregulation of TGF-beta receptor signaling / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / 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 / branch elongation involved in mammary gland duct branching / positive regulation of primary miRNA processing / columnar/cuboidal epithelial cell maturation / negative regulation of skeletal muscle tissue development / response to laminar fluid shear stress / embryonic liver development / regulation of branching involved in mammary gland duct morphogenesis / regulation of cartilage development / regulation of striated muscle tissue development / Regulation of RUNX3 expression and activity / regulation of protein import into nucleus / tolerance induction to self antigen / cellular response to acetaldehyde / negative regulation of natural killer cell mediated cytotoxicity directed against tumor cell target / negative regulation of hyaluronan biosynthetic process / extracellular matrix assembly / type III transforming growth factor beta receptor binding / positive regulation of odontogenesis / positive regulation of smooth muscle cell differentiation / positive regulation of exit from mitosis / secondary palate development / negative regulation of macrophage cytokine production / mononuclear cell proliferation / odontoblast differentiation / Cell surface interactions at the vascular wall / positive regulation of isotype switching to IgA isotypes / positive regulation of mesenchymal stem cell proliferation / positive regulation of receptor signaling pathway via STAT / membrane protein intracellular domain proteolysis / retina vasculature development in camera-type eye / positive regulation of extracellular matrix assembly / heart valve morphogenesis / T-helper 17 cell lineage commitment / mammary gland branching involved in thelarche / regulation of regulatory T cell differentiation / Platelet degranulation / positive regulation of vasculature development / hyaluronan catabolic process / lens fiber cell differentiation / ATP biosynthetic process / cell activation / type II transforming growth factor beta receptor binding / positive regulation of branching involved in ureteric bud morphogenesis / receptor catabolic process / transforming growth factor beta receptor binding / positive regulation of cardiac muscle cell differentiation / response to salt / receptor ligand inhibitor activity / germ cell migration / regulatory T cell differentiation / endoderm development / negative regulation of cell-cell adhesion mediated by cadherin / positive regulation of chemotaxis / type I transforming growth factor beta receptor binding / negative regulation of myoblast differentiation / phospholipid homeostasis / positive regulation of mononuclear cell migration / positive regulation of vascular permeability / myeloid dendritic cell differentiation / negative regulation of biomineral tissue development / mammary gland development / oligodendrocyte development / negative regulation of interleukin-17 production / deubiquitinase activator activity / phosphate-containing compound metabolic process / surfactant homeostasis / cell-cell junction organization / response to cholesterol / transforming growth factor beta binding / sprouting angiogenesis / positive regulation of chemokine (C-X-C motif) ligand 2 production / negative regulation of ossification / negative regulation of release of sequestered calcium ion into cytosol / aortic valve morphogenesis / digestive tract development / response to vitamin D / face morphogenesis / positive regulation of fibroblast migration / ureteric bud development / neural tube development / positive regulation of regulatory T cell differentiation / negative regulation of neuroblast proliferation / negative regulation of cytokine production
Similarity search - Function
Transforming growth factor beta-1 proprotein / Transforming growth factor-beta / 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 ...Transforming growth factor beta-1 proprotein / Transforming growth factor-beta / 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, 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 activator LRRC32 / Transforming growth factor beta-1 proprotein
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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
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 1.0Jun 25, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 25, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 25, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 25, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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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 proprotein
B: Transforming growth factor beta-1 proprotein
C: Transforming growth factor beta-1 proprotein
D: Transforming growth factor beta-1 proprotein
E: Transforming growth factor beta activator LRRC32
F: mFab LMT-12, Light Chain
G: mFab LMT-12, Heavy Chain
H: mFab LMT-12, Heavy Chain
I: mFab LMT-12, Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)354,60914
Polymers352,8939
Non-polymers1,7165
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23250 Å2
ΔGint-80 kcal/mol
Surface area64510 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 proprotein


Mass: 44369.926 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Tgfb1 / Production host: Homo sapiens (human) / References: UniProt: P04202
#2: Protein Transforming growth factor beta activator LRRC32 / Garpin / Glycoprotein A repetitions predominant / GARP / Leucine-rich repeat-containing protein 32


Mass: 73699.914 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc32 / Production host: Homo sapiens (human) / References: UniProt: G3XA59

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Antibody , 2 types, 4 molecules FIGH

#3: Antibody mFab LMT-12, Light Chain


Mass: 24713.555 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#4: Antibody mFab LMT-12, Heavy Chain


Mass: 26143.363 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)

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Sugars , 2 types, 5 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.: 3
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: 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.: 2
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
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: mouse GAPR-lTGFbeta1 in complex with Fab LMT-12. / Type: COMPLEX / Entity ID: #2-#4 / Source: RECOMBINANT
Molecular weightValue: 0.2599 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.12 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: 1 / Num. of real images: 13898
Details: A total of 6605 untilted movies were collected followed by 3508 movies at 20 degree tilt.

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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: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 288887 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11AF-G3XA59-F11AlphaFoldin silico model
21AF-P040202-F12AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412626
ELECTRON MICROSCOPYf_angle_d0.74617161
ELECTRON MICROSCOPYf_dihedral_angle_d4.9861764
ELECTRON MICROSCOPYf_chiral_restr0.0461963
ELECTRON MICROSCOPYf_plane_restr0.0062205

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