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- PDB-8cr6: mouse Interleukin-12 -

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Basic information

Entry
Database: PDB / ID: 8cr6
Titlemouse Interleukin-12
Components
  • Interleukin-12 subunit alpha
  • Interleukin-12 subunit beta
KeywordsCYTOKINE / inflammation / immunity / heterodimer
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 / positive regulation of smooth muscle cell apoptotic process / positive regulation of T-helper 1 type immune response / interleukin-12 receptor binding / T-helper cell differentiation / interleukin-12-mediated signaling pathway / positive regulation of NK T cell proliferation / negative regulation of interleukin-17 production / positive regulation of osteoclast differentiation / cytokine receptor activity / natural killer cell activation / positive regulation of granulocyte macrophage colony-stimulating factor production / response to UV-B / positive regulation of T cell differentiation / negative regulation of interleukin-10 production / defense response to protozoan / positive regulation of interleukin-17 production / positive regulation of natural killer cell proliferation / positive regulation of activated T cell proliferation / positive regulation of interleukin-10 production / negative regulation of protein secretion / cell surface receptor signaling pathway via JAK-STAT / T cell proliferation / positive regulation of defense response to virus by host / positive regulation of T cell proliferation / positive regulation of interleukin-12 production / endosome lumen / cytokine activity / growth factor activity / negative regulation of smooth muscle cell proliferation / negative regulation of inflammatory response to antigenic stimulus / cellular response to type II interferon / positive regulation of T cell mediated cytotoxicity / Golgi lumen / positive regulation of type II interferon production / positive regulation of tumor necrosis factor production / cell migration / cellular response to lipopolysaccharide / defense response to Gram-negative bacterium / defense response to virus / cell surface receptor signaling pathway / cell population proliferation / immune response / endoplasmic reticulum lumen / protein heterodimerization activity / cell surface / extracellular space / extracellular region / identical protein binding / membrane / cytoplasm
Similarity search - Function
Interleukin-12 alpha / Interleukin-12 alpha subunit / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. / Four-helical cytokine-like, core / Immunoglobulin subtype 2 ...Interleukin-12 alpha / Interleukin-12 alpha subunit / Interleukin-12 beta / Interleukin-12 beta, central domain / : / Cytokine interleukin-12p40 C-terminus / Long hematopoietin receptor, soluble alpha chain, conserved site / Long hematopoietin receptor, soluble alpha chains family signature. / Four-helical cytokine-like, core / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Fibronectin type III / Fibronectin type III superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Interleukin-12 subunit alpha / Interleukin-12 subunit beta
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.85 Å
AuthorsMerceron, R. / Felix, J. / Lambert, E. / 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 7, 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 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _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 beta
B: Interleukin-12 subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,4944
Polymers66,6862
Non-polymers8082
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering, same assembly as previously reported for human ortholog
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3270 Å2
ΔGint-3 kcal/mol
Surface area24100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.110, 70.770, 127.370
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Interleukin-12 subunit beta / IL-12B / Cytotoxic lymphocyte maturation factor 40 kDa subunit / CLMF p40 / IL-12 subunit p40


Mass: 38282.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: [M1-A22] encode the signal peptide and are most likely removed during translation/folding.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12b / Plasmid: pHLsec / Cell line (production host): HEK293S MGAT1-/- / Production host: Homo sapiens (human) / References: UniProt: P43432
#2: Protein Interleukin-12 subunit alpha / IL-12A / Cytotoxic lymphocyte maturation factor 35 kDa subunit / CLMF p35 / IL-12 subunit p35


Mass: 28403.510 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues [1-22] encode the signal peptide which is most likely removed during translation/folding. Residues [216-253] encode cloning scar, protease site, avi-tag and his-tag. Residues [224- ...Details: Residues [1-22] encode the signal peptide which is most likely removed during translation/folding. Residues [216-253] encode cloning scar, protease site, avi-tag and his-tag. Residues [224-253] are most likely removed due to protease treatment.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Il12a / Plasmid: pHLsec / Cell line (production host): HEK293S MGAT1-/- / Production host: Homo sapiens (human) / References: UniProt: P43431
#3: 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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#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.: 1 / 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
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: 2 Å3/Da / Density % sol: 38.42 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: JCSG-plus G4: 0.2 M TMAO, 0.1 M Tris pH 8.5, 20% w/v/ PEG 2000 MME, 20 mM BaCl2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 1.008 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 22, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.008 Å / Relative weight: 1
ReflectionResolution: 2.85→47.34 Å / Num. obs: 12530 / % possible obs: 96.1 % / Redundancy: 2.91 % / Biso Wilson estimate: 97.92 Å2 / CC1/2: 0.999 / Rrim(I) all: 0.058 / Net I/σ(I): 13.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
8.43-47.342.5835.85210.9980.03291.2
6.01-8.432.7631.758700.9980.03593.4
4.92-6.012.8928.4211100.9980.04195.3
4.26-4.922.8824.2512980.9970.04995.5
3.82-4.262.9918.2414860.9940.07196.4
3.49-3.822.9311.4916180.9870.11897.3
3.23-3.492.956.3417480.9660.22596.2
3.02-3.233.023.0119040.8620.4897.8
2.85-3.022.881.219750.5751.0996.4

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDS20170215data reduction
XSCALE20170215data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.85→47.34 Å / SU ML: 0.5429 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 36.5964
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2956 627 5.01 %
Rwork0.2545 11900 -
obs0.2567 12527 96.22 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 110.55 Å2
Refinement stepCycle: LAST / Resolution: 2.85→47.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3584 0 53 2 3639
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00253704
X-RAY DIFFRACTIONf_angle_d0.49355016
X-RAY DIFFRACTIONf_chiral_restr0.0373584
X-RAY DIFFRACTIONf_plane_restr0.0029633
X-RAY DIFFRACTIONf_dihedral_angle_d11.17281368
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.85-3.140.47921550.392951X-RAY DIFFRACTION97.55
3.14-3.590.3531550.30572933X-RAY DIFFRACTION96.8
3.59-4.520.28081560.25842974X-RAY DIFFRACTION96.28
4.53-47.340.26771610.22363042X-RAY DIFFRACTION94.37
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.51504571437-2.670307850950.7439585340474.778332218810.6002123055362.18990118154-0.194605191438-0.321372547673-0.3951834992090.2110944865910.4014978502640.07146608329150.7196649198190.04955970875810.0164070292520.8126762414250.03694081522950.1279483083830.6689322062720.07009873017210.565292820595-33.4308432333-9.2023827110317.6419515327
26.89716304167-1.50940021843-1.387161349634.34885484705-2.791769822775.96130731482-0.4141327608520.3403075022670.632887646198-0.04365657475160.0284465847272-0.596176971822-0.483529319975-0.239087626021-0.01847584723060.612400515807-0.105879383176-0.01121239274110.525001612737-0.04551245940770.843223030915-35.343969955821.64450680737.42174148273
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-IDEnd label seq-ID
11(chain 'A' )AA - D23 - 3521
22(chain 'B' )BB - E1 - 215165

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