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- PDB-8c7h: Cryo-EM Map of the latTGF-beta 28G11 Fab complex -

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Basic information

Entry
Database: PDB / ID: 8c7h
TitleCryo-EM Map of the latTGF-beta 28G11 Fab complex
Components
  • (Transforming growth factor beta- ...) x 2
  • 28G11 Fab heavy chain
  • 28G11 Fab light chain
  • Transforming growth factor beta activator LRRC32
KeywordsIMMUNE SYSTEM / Fab-complex / GARP / lat-TGF-beta
Function / homology
Function and homology information


: / positive regulation of primary miRNA processing / positive regulation of microglia differentiation / Influenza Virus Induced Apoptosis / negative regulation of skeletal muscle tissue development / TGFBR2 MSI Frameshift Mutants in Cancer / regulatory T cell differentiation / regulation of blood vessel remodeling / regulation of striated muscle tissue development / negative regulation of natural killer cell mediated cytotoxicity directed against tumor cell target ...: / positive regulation of primary miRNA processing / positive regulation of microglia differentiation / Influenza Virus Induced Apoptosis / negative regulation of skeletal muscle tissue development / TGFBR2 MSI Frameshift Mutants in Cancer / regulatory T cell differentiation / regulation of blood vessel remodeling / regulation of striated muscle tissue development / negative regulation of natural killer cell mediated cytotoxicity directed against tumor cell target / regulation of protein import into nucleus / extracellular matrix assembly / embryonic liver development / type III transforming growth factor beta receptor binding / negative regulation of hyaluronan biosynthetic process / positive regulation of cardiac muscle cell differentiation / myofibroblast differentiation / connective tissue replacement involved in inflammatory response wound healing / odontoblast differentiation / negative regulation of macrophage cytokine production / positive regulation of receptor signaling pathway via STAT / TGFBR2 Kinase Domain Mutants in Cancer / : / positive regulation of isotype switching to IgA isotypes / positive regulation of mesenchymal stem cell proliferation / secondary palate development / membrane protein intracellular domain proteolysis / SMAD2/3 Phosphorylation Motif Mutants in Cancer / TGFBR1 KD Mutants in Cancer / heart valve morphogenesis / positive regulation of vasculature development / hyaluronan catabolic process / regulation of transforming growth factor beta receptor signaling pathway / ATP biosynthetic process / receptor catabolic process / negative regulation of extracellular matrix disassembly / positive regulation of extracellular matrix assembly / type II transforming growth factor beta receptor binding / TGFBR1 LBD Mutants in Cancer / positive regulation of chemotaxis / negative regulation of biomineral tissue development / type I transforming growth factor beta receptor binding / cell-cell junction organization / negative regulation of myoblast differentiation / positive regulation of vascular permeability / deubiquitinase activator activity / transforming growth factor beta binding / response to cholesterol / positive regulation of endothelial cell apoptotic process / positive regulation of chemokine (C-X-C motif) ligand 2 production / aortic valve morphogenesis / positive regulation of fibroblast migration / phosphate-containing compound metabolic process / negative regulation of protein localization to plasma membrane / sprouting angiogenesis / neural tube development / Molecules associated with elastic fibres / RUNX3 regulates CDKN1A transcription / negative regulation of cytokine production / positive regulation of epidermal growth factor receptor signaling pathway / ventricular cardiac muscle tissue morphogenesis / macrophage derived foam cell differentiation / negative regulation of fat cell differentiation / Syndecan interactions / negative regulation of cell-cell adhesion / positive regulation of interleukin-17 production / TGF-beta receptor signaling activates SMADs / plasma membrane => GO:0005886 / negative regulation of activated T cell proliferation / negative regulation of cell differentiation / positive regulation of SMAD protein signal transduction / RUNX3 regulates p14-ARF / positive regulation of cell division / cellular response to low-density lipoprotein particle stimulus / negative regulation of blood vessel endothelial cell migration / negative regulation of cell cycle / ECM proteoglycans / positive regulation of vascular endothelial growth factor production / positive regulation of collagen biosynthetic process / epithelial to mesenchymal transition / positive regulation of epithelial to mesenchymal transition / positive regulation of blood vessel endothelial cell migration / vasculogenesis / lymph node development / chondrocyte differentiation / hematopoietic progenitor cell differentiation / salivary gland morphogenesis / extrinsic apoptotic signaling pathway / positive regulation of protein dephosphorylation / regulation of cell migration / cellular response to transforming growth factor beta stimulus / antigen binding / positive regulation of protein metabolic process / protein export from nucleus / Downregulation of TGF-beta receptor signaling / extracellular matrix / transforming growth factor beta receptor signaling pathway / positive regulation of superoxide anion generation / negative regulation of miRNA transcription / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition)
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 / Leucine-rich repeat N-terminal domain / Leucine rich repeat N-terminal domain ...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 / Leucine-rich repeat N-terminal domain / Leucine rich repeat N-terminal domain / Transforming growth factor-beta (TGF-beta) family / Transforming growth factor-beta, C-terminal / Transforming growth factor beta like domain / TGF-beta family profile. / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsEbenhoch, R. / Nar, H.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Immunohorizons / Year: 2023
Title: Anti-GARP Antibodies Inhibit Release of TGF-β by Regulatory T Cells via Different Modes of Action, but Do Not Influence Their Function In Vitro.
Authors: Frederik H Igney / Rebecca Ebenhoch / Felix Schiele / Herbert Nar /
Abstract: Regulatory T cells (Treg) play a critical role in controlling immune responses in diseases such as cancer or autoimmunity. Activated Treg express the membrane protein GARP (LRRC32) in complex with ...Regulatory T cells (Treg) play a critical role in controlling immune responses in diseases such as cancer or autoimmunity. Activated Treg express the membrane protein GARP (LRRC32) in complex with the latent form of the immunosuppressive cytokine TGF-β (L-TGF-β). In this study, we confirmed that active TGF-β was generated from its latent form in an integrin-dependent manner and induced TGF-β receptor signaling in activated human Treg. We studied a series of Abs targeting the L-TGF-β/GARP complex with distinct binding modes. We found that TGF-β receptor signaling could be inhibited by anti-TGF-β and by some, but not all, Abs against the L-TGF-β/GARP complex. Cryogenic electron microscopy structures of three L-TGF-β/GARP complex-targeting Abs revealed their distinct epitopes and allowed us to elucidate how they achieve blockade of TGF-β activation. Three different modes of action were identified, including a novel unusual mechanism of a GARP-binding Ab. However, blockade of GARP or TGF-β by Abs did not influence the suppressive activity of human Treg in vitro. We were also not able to confirm a prominent role of GARP in other functions of human Treg, such as FOXP3 induction and Treg stability. These data show that the GARP/TGF-β axis can be targeted pharmacologically in different ways, but further studies are necessary to understand its complexity and to unleash its therapeutic potential.
History
DepositionJan 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._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 / _citation_author.identifier_ORCID / _citation_author.name

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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
I: Transforming growth factor beta activator LRRC32
L: 28G11 Fab heavy chain
K: 28G11 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,42210
Polymers195,6627
Non-polymers1,7603
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18620 Å2
ΔGint-68 kcal/mol
Surface area57000 Å2

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Components

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Transforming growth factor beta- ... , 2 types, 4 molecules ACBD

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


Mass: 28531.488 Da / Num. of mol.: 2 / 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-1 / / TGF-beta-1


Mass: 12809.812 Da / Num. of mol.: 2 / Fragment: Mature
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

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Antibody , 2 types, 2 molecules LK

#4: Antibody 28G11 Fab heavy chain


Mass: 24529.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody 28G11 Fab light chain


Mass: 23091.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Protein / Sugars , 2 types, 4 molecules I

#3: Protein Transforming growth factor beta activator LRRC32 / Garpin / Glycoprotein A repetitions predominant / GARP / Leucine-rich repeat-containing protein 32


Mass: 65358.488 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
#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.: 3
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

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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: latTGF-beta in complex with Fab 28G11 / Type: COMPLEX / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_4839: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 418886 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411889
ELECTRON MICROSCOPYf_angle_d0.67916246
ELECTRON MICROSCOPYf_dihedral_angle_d4.4621750
ELECTRON MICROSCOPYf_chiral_restr0.0421944
ELECTRON MICROSCOPYf_plane_restr0.0092095

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