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- PDB-8ynm: Structure of the Caspase-8/cFLIP death effector domain assembly -

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Basic information

Entry
Database: PDB / ID: 8ynm
TitleStructure of the Caspase-8/cFLIP death effector domain assembly
Components
  • CASP8 and FADD-like apoptosis regulator subunit p43
  • Caspase-8 subunit p10
KeywordsAPOPTOSIS / FADD / caspase-8 / cellular FLICE-like inhibitory protein / Death effector domain
Function / homology
Function and homology information


negative regulation of myoblast fusion / skeletal myofibril assembly / caspase-8 / death effector domain binding / FasL/ CD95L signaling / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / skeletal muscle atrophy / TRAIL signaling / CD95 death-inducing signaling complex / regulation of skeletal muscle satellite cell proliferation ...negative regulation of myoblast fusion / skeletal myofibril assembly / caspase-8 / death effector domain binding / FasL/ CD95L signaling / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / skeletal muscle atrophy / TRAIL signaling / CD95 death-inducing signaling complex / regulation of skeletal muscle satellite cell proliferation / ripoptosome / Defective RIPK1-mediated regulated necrosis / TRAIL-activated apoptotic signaling pathway / Apoptotic execution phase / Activation, myristolyation of BID and translocation to mitochondria / TRIF-mediated programmed cell death / Microbial modulation of RIPK1-mediated regulated necrosis / Regulation by c-FLIP / CASP8 activity is inhibited / Dimerization of procaspase-8 / TLR3-mediated TICAM1-dependent programmed cell death / regulation of necroptotic process / Caspase activation via Death Receptors in the presence of ligand / positive regulation of extracellular matrix organization / positive regulation of macrophage differentiation / self proteolysis / positive regulation of glomerular mesangial cell proliferation / response to cobalt ion / NF-kB activation through FADD/RIP-1 pathway mediated by caspase-8 and -10 / death-inducing signaling complex / skeletal muscle tissue regeneration / CLEC7A/inflammasome pathway / negative regulation of necroptotic process / response to anesthetic / negative regulation of hepatocyte apoptotic process / regulation of tumor necrosis factor-mediated signaling pathway / tumor necrosis factor receptor binding / death receptor binding / positive regulation of hepatocyte proliferation / natural killer cell activation / negative regulation of cellular response to transforming growth factor beta stimulus / TNFR1-induced proapoptotic signaling / RIPK1-mediated regulated necrosis / negative regulation of cardiac muscle cell apoptotic process / execution phase of apoptosis / regulation of innate immune response / Apoptotic cleavage of cellular proteins / pyroptotic inflammatory response / response to testosterone / : / B cell activation / positive regulation of proteolysis / macrophage differentiation / response to tumor necrosis factor / extrinsic apoptotic signaling pathway via death domain receptors / Caspase-mediated cleavage of cytoskeletal proteins / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / skeletal muscle tissue development / negative regulation of reactive oxygen species biosynthetic process / extrinsic apoptotic signaling pathway / negative regulation of canonical NF-kappaB signal transduction / cellular response to nitric oxide / cysteine-type peptidase activity / cellular response to epidermal growth factor stimulus / regulation of cytokine production / cellular response to dexamethasone stimulus / protein maturation / proteolysis involved in protein catabolic process / T cell activation / enzyme activator activity / positive regulation of interleukin-1 beta production / negative regulation of extrinsic apoptotic signaling pathway / Regulation of NF-kappa B signaling / apoptotic signaling pathway / cellular response to estradiol stimulus / Regulation of TNFR1 signaling / wound healing / NOD1/2 Signaling Pathway / protein processing / positive regulation of neuron projection development / Regulation of necroptotic cell death / cellular response to insulin stimulus / cellular response to mechanical stimulus / positive regulation of NF-kappaB transcription factor activity / positive regulation of neuron apoptotic process / response to estradiol / lamellipodium / peptidase activity / heart development / cell body / protease binding / scaffold protein binding / angiogenesis / cellular response to hypoxia / response to lipopolysaccharide / response to ethanol / mitochondrial outer membrane / positive regulation of canonical NF-kappaB signal transduction / cytoskeleton / positive regulation of ERK1 and ERK2 cascade
Similarity search - Function
Caspase-8 / Death effector domain / Death effector domain (DED) profile. / Death effector domain / Death effector domain / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. ...Caspase-8 / Death effector domain / Death effector domain (DED) profile. / Death effector domain / Death effector domain / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain / Caspase family p20 domain profile. / : / Caspase domain / Caspase-like domain superfamily / Death-like domain superfamily
Similarity search - Domain/homology
CASP8 and FADD-like apoptosis regulator / Caspase-8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsLin, S.-C. / Yang, C.-Y.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Other government Taiwan
CitationJournal: Nat Commun / Year: 2024
Title: Reverse hierarchical DED assembly in the cFLIP-procaspase-8 and cFLIP-procaspase-8-FADD complexes.
Authors: Chao-Yu Yang / Yi-Chun Tseng / Yi-Fan Tu / Bai-Jiun Kuo / Li-Chung Hsu / Chia-I Lien / You-Sheng Lin / Yin-Ting Wang / Yen-Chen Lu / Tsung-Wei Su / Yu-Chih Lo / Su-Chang Lin /
Abstract: cFLIP, a master anti-apoptotic regulator, targets the FADD-induced DED complexes of procaspase-8 in death receptor and ripoptosome signaling pathways. Several tumor cells maintain relatively high ...cFLIP, a master anti-apoptotic regulator, targets the FADD-induced DED complexes of procaspase-8 in death receptor and ripoptosome signaling pathways. Several tumor cells maintain relatively high levels of cFLIP in achieving their immortality. However, understanding the three-dimensional regulatory mechanism initiated or mediated by elevated levels of cFLIP has been limited by the absence of the atomic coordinates for cFLIP-induced DED complexes. Here we report the crystal plus cryo-EM structures to uncover an unconventional mechanism where cFLIP and procaspase-8 autonomously form a binary tandem DED complex, independent of FADD. This complex gains the ability to recruit FADD, thereby allosterically modulating cFLIP assembly and partially activating caspase-8 for RIPK1 cleavage. Our structure-guided mutagenesis experiments provide critical insights into these regulatory mechanisms, elucidating the resistance to apoptosis and necroptosis in achieving immortality. Finally, this research offers a unified model for the intricate bidirectional hierarchy-based processes using multiprotein helical assembly to govern cell fate decisions.
History
DepositionMar 11, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
O: CASP8 and FADD-like apoptosis regulator subunit p43
A: Caspase-8 subunit p10
B: Caspase-8 subunit p10
C: Caspase-8 subunit p10
H: CASP8 and FADD-like apoptosis regulator subunit p43
I: CASP8 and FADD-like apoptosis regulator subunit p43
J: CASP8 and FADD-like apoptosis regulator subunit p43
K: CASP8 and FADD-like apoptosis regulator subunit p43
G: CASP8 and FADD-like apoptosis regulator subunit p43
F: CASP8 and FADD-like apoptosis regulator subunit p43
N: CASP8 and FADD-like apoptosis regulator subunit p43


Theoretical massNumber of molelcules
Total (without water)332,60311
Polymers332,60311
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CASP8 and FADD-like apoptosis regulator subunit p43


Mass: 20878.479 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CFLAR, CASH, CASP8AP1, CLARP, MRIT / Production host: Escherichia coli (E. coli) / References: UniProt: O15519
#2: Protein Caspase-8 subunit p10


Mass: 55191.648 Da / Num. of mol.: 3 / Mutation: F122G, L123G, C360A, D374A, D384A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CASP8, MCH5 / Production host: Escherichia coli (E. coli) / References: UniProt: Q14790
Has protein modificationN

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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: Caspase-8/cFLIP death effector domain assembly / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMsodium chlorideNaCl1
220 mMTris(HOCH2)3CNH21
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 279 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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 72 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 80

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487:model refinement
3EPUimage acquisition
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 246000
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28464 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215821
ELECTRON MICROSCOPYf_angle_d0.37621195
ELECTRON MICROSCOPYf_dihedral_angle_d3.1932089
ELECTRON MICROSCOPYf_chiral_restr0.0342484
ELECTRON MICROSCOPYf_plane_restr0.0032682

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