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- PDB-8oe0: Cryo-EM structure of a pre-dimerized murine IL-12 complete extrac... -

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Basic information

Entry
Database: PDB / ID: 8oe0
TitleCryo-EM structure of a pre-dimerized murine IL-12 complete extracellular signaling complex (Class 2).
Components
  • (Interleukin-12 receptor subunit beta- ...) x 2
  • (Interleukin-12 subunit ...) x 2
KeywordsSIGNALING PROTEIN / Complex / Cytokine / Receptor
Function / homology
Function and homology information


interleukin-12 beta subunit binding / Interleukin-12 signaling / Interleukin-23 signaling / interleukin-23 receptor binding / Interleukin-35 Signalling / interleukin-12 alpha subunit binding / interleukin-12 complex / interleukin-23 complex / T-helper 1 cell activation / negative regulation of vascular endothelial growth factor signaling pathway ...interleukin-12 beta subunit binding / Interleukin-12 signaling / Interleukin-23 signaling / interleukin-23 receptor binding / Interleukin-35 Signalling / interleukin-12 alpha subunit binding / interleukin-12 complex / interleukin-23 complex / T-helper 1 cell activation / negative regulation of vascular endothelial growth factor signaling pathway / positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target / negative regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / cellular response to hydroperoxide / positive regulation of smooth muscle cell apoptotic process / regulation of response to tumor cell / positive regulation of autophagic cell death / DAPK1-calmodulin complex / positive regulation of T-helper 1 type immune response / interleukin-12 receptor binding / interleukin-23 receptor complex / T-helper cell differentiation / Caspase activation via Dependence Receptors in the absence of ligand / interleukin-12-mediated signaling pathway / defense response to tumor cell / positive regulation of NK T cell proliferation / regulation of NMDA receptor activity / negative regulation of interleukin-17 production / positive regulation of osteoclast differentiation / calcium/calmodulin-dependent protein kinase activity / cytokine receptor activity / natural killer cell activation / positive regulation of granulocyte macrophage colony-stimulating factor production / response to UV-B / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / syntaxin-1 binding / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / positive regulation of T cell differentiation / CREB1 phosphorylation through the activation of Adenylate Cyclase / CaMK IV-mediated phosphorylation of CREB / PKA activation / negative regulation of high voltage-gated calcium channel activity / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / Activation of RAC1 downstream of NMDARs / negative regulation of interleukin-10 production / negative regulation of ryanodine-sensitive calcium-release channel activity / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / defense response to protozoan / autophagosome membrane docking / negative regulation of calcium ion export across plasma membrane / regulation of cardiac muscle cell action potential / presynaptic endocytosis / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / Phase 0 - rapid depolarisation / calcineurin-mediated signaling / Negative regulation of NMDA receptor-mediated neuronal transmission / positive regulation of interleukin-17 production / positive regulation of natural killer cell proliferation / positive regulation of activated T cell proliferation / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / positive regulation of interleukin-10 production / regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / Long-term potentiation / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / extrinsic apoptotic signaling pathway via death domain receptors / DARPP-32 events / catalytic complex / Smooth Muscle Contraction / negative regulation of protein secretion / detection of calcium ion / cell surface receptor signaling pathway via JAK-STAT / regulation of cardiac muscle contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / T cell proliferation / cellular response to interferon-beta / Protein methylation / calcium channel inhibitor activity / coreceptor activity / Activation of AMPK downstream of NMDARs / presynaptic cytosol / Ion homeostasis / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / titin binding / positive regulation of defense response to virus by host / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation
Similarity search - Function
Interleukin-12 alpha / Interleukin-12 alpha subunit / : / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Death-associated protein kinase 1 / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. ...Interleukin-12 alpha / Interleukin-12 alpha subunit / : / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Death-associated protein kinase 1 / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Roc domain profile. / Roc domain / Ankyrin repeats (many copies) / Death domain profile. / DEATH domain, found in proteins involved in cell death (apoptosis). / Death domain / Death domain / Four-helical cytokine-like, core / : / Death-like domain superfamily / Ankyrin repeat / Fibronectin type III domain / Fibronectin type 3 domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / EF-hand domain pair / Fibronectin type-III domain profile. / Fibronectin type III / Ankyrin repeats (3 copies) / Fibronectin type III superfamily / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / EF-hand, calcium binding motif / Ankyrin repeat-containing domain superfamily / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Calmodulin-1 / Interleukin-12 subunit alpha / Interleukin-12 subunit beta / Death-associated protein kinase 1 / Interleukin-12 receptor subunit beta-2 / Interleukin-12 receptor subunit beta-1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsFelix, J. / Bloch, Y. / Savvides, S.N.
Funding support Belgium, 2items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)12S0519N Belgium
Research Foundation - Flanders (FWO)G0B4918N Belgium
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23.
Authors: Yehudi Bloch / Jan Felix / Romain Merceron / Mathias Provost / Royan Alipour Symakani / Robin De Backer / Elisabeth Lambert / Ahmad R Mehdipour / Savvas N Savvides /
Abstract: Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into ...Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12-receptor interaction interfaces, in contrast to IL-23-receptor complexes. Here we report structures of fully assembled mouse IL-12/human IL-23-receptor complexes comprising the complete extracellular segments of the cognate receptors determined by electron cryo-microscopy. The structures reveal key commonalities but also surprisingly diverse features. Most notably, whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig domain of their high-affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell membrane. Collectively, our findings will help to complete our understanding of cytokine-mediated assemblies of tall cytokine receptors and will enable a cytokine-specific interrogation of IL-12/IL-23 signaling in physiology and disease.
History
DepositionMar 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Interleukin-12 subunit alpha
B: Interleukin-12 subunit beta
C: Interleukin-12 receptor subunit beta-1,Death-associated protein kinase 1
D: Interleukin-12 receptor subunit beta-2,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)212,9529
Polymers211,2774
Non-polymers1,6755
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, light scattering, MALLS, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Interleukin-12 subunit ... , 2 types, 2 molecules AB

#1: Protein Interleukin-12 subunit alpha / IL-12A / Cytotoxic lymphocyte maturation factor 35 kDa subunit / CLMF p35 / IL-12 subunit p35


Mass: 25927.496 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12a / Cell line (production host): HEK293 MGAT-/- / Production host: Homo sapiens (human) / References: UniProt: P43431
#2: Protein Interleukin-12 subunit beta / IL-12B / Cytotoxic lymphocyte maturation factor 40 kDa subunit / CLMF p40 / IL-12 subunit p40


Mass: 35837.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12b / Cell line (production host): HEK293 MGAT-/- / Production host: Homo sapiens (human) / References: UniProt: P43432

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Interleukin-12 receptor subunit beta- ... , 2 types, 2 molecules CD

#3: Protein Interleukin-12 receptor subunit beta-1,Death-associated protein kinase 1 / IL-12 receptor subunit beta-1 / IL-12R subunit beta-1 / IL-12R-beta-1 / IL-12 receptor beta ...IL-12 receptor subunit beta-1 / IL-12R subunit beta-1 / IL-12R-beta-1 / IL-12 receptor beta component / DAP kinase 1


Mass: 63789.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12rb1, Il12rb, DAPK1, DAPK / Production host: Homo sapiens (human) / Strain (production host): HEK293 MGAT-/-
References: UniProt: Q60837, UniProt: P53355, non-specific serine/threonine protein kinase
#4: Protein Interleukin-12 receptor subunit beta-2,Calmodulin-1 / IL-12 receptor subunit beta-2 / IL-12R subunit beta-2 / IL-12R-beta-2 / IL-12RB2


Mass: 85723.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12rb2, CALM1, CALM, CAM, CAM1 / Cell line (production host): HEK293 MGAT-/- / Production host: Homo sapiens (human) / References: UniProt: P97378, UniProt: P0DP23

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Sugars , 3 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.: 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
#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.: 1
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
#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.: 3
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: Murine IL-12 in complex with mIL-12Rbeta1-DAPK1 and mIL-12Rbeta2-Calmodulin.
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.216 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Details: HEPES-buffered saline (HBS) with added calcium chloride: 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
225 mMHEPESC8H18N2O4S1
35 mMcalcium chlorideCaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Details: Leica EM GP2, 5 s. blotting time.

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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: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.37 sec. / Electron dose: 61.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8145

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.19.2-4158model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2151468
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209938 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 328.9 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004712261
ELECTRON MICROSCOPYf_angle_d0.922616660
ELECTRON MICROSCOPYf_chiral_restr0.05691874
ELECTRON MICROSCOPYf_plane_restr0.00762122
ELECTRON MICROSCOPYf_dihedral_angle_d14.41124532

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