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- PDB-6xkj: Cryo-EM structure of CARD8-CARD filament -

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Basic information

Entry
Database: PDB / ID: 6xkj
TitleCryo-EM structure of CARD8-CARD filament
ComponentsCaspase recruitment domain-containing protein 8
KeywordsIMMUNE SYSTEM / Filament / inflammasome / signaling / UPA / FIIND / CARD / NLRP1
Function / homology
Function and homology information


CARD8 inflammasome complex assembly / NACHT domain binding / Formation of apoptosome / cysteine-type endopeptidase activator activity / inhibition of cysteine-type endopeptidase activity / negative regulation of NLRP3 inflammasome complex assembly / NLRP3 inflammasome complex / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway / self proteolysis ...CARD8 inflammasome complex assembly / NACHT domain binding / Formation of apoptosome / cysteine-type endopeptidase activator activity / inhibition of cysteine-type endopeptidase activity / negative regulation of NLRP3 inflammasome complex assembly / NLRP3 inflammasome complex / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway / self proteolysis / Regulation of the apoptosome activity / Hydrolases; Acting on peptide bonds (peptidases) / regulation of canonical NF-kappaB signal transduction / pattern recognition receptor activity / negative regulation of interleukin-1 beta production / negative regulation of NF-kappaB transcription factor activity / antiviral innate immune response / cysteine-type endopeptidase activator activity involved in apoptotic process / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of canonical NF-kappaB signal transduction / molecular condensate scaffold activity / positive regulation of interleukin-1 beta production / peptidase activity / defense response to virus / regulation of apoptotic process / protein homodimerization activity / protein-containing complex / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
FIIND domain / Function to find / FIIND domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Caspase recruitment domain-containing protein 8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsHollingsworth, L.R. / David, L. / Li, Y. / Sharif, H. / Fontana, P. / Fu, T. / Wu, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director1DP1 HD087988 United States
CitationJournal: Nat Commun / Year: 2021
Title: Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.
Authors: L Robert Hollingsworth / Liron David / Yang Li / Andrew R Griswold / Jianbin Ruan / Humayun Sharif / Pietro Fontana / Elizabeth L Orth-He / Tian-Min Fu / Daniel A Bachovchin / Hao Wu /
Abstract: NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation ...NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1 filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASC-caspase-1 octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.
History
DepositionJun 26, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 25, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _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
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
C: Caspase recruitment domain-containing protein 8
A: Caspase recruitment domain-containing protein 8
B: Caspase recruitment domain-containing protein 8
D: Caspase recruitment domain-containing protein 8
E: Caspase recruitment domain-containing protein 8
F: Caspase recruitment domain-containing protein 8
G: Caspase recruitment domain-containing protein 8
H: Caspase recruitment domain-containing protein 8
I: Caspase recruitment domain-containing protein 8
J: Caspase recruitment domain-containing protein 8
K: Caspase recruitment domain-containing protein 8
L: Caspase recruitment domain-containing protein 8
M: Caspase recruitment domain-containing protein 8
N: Caspase recruitment domain-containing protein 8
O: Caspase recruitment domain-containing protein 8
P: Caspase recruitment domain-containing protein 8


Theoretical massNumber of molelcules
Total (without water)161,65416
Polymers161,65416
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23190 Å2
ΔGint-1 kcal/mol
Surface area61710 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 16 / Rise per n subunits: 5.2 Å / Rotation per n subunits: -99.07 °)

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Components

#1: Protein
Caspase recruitment domain-containing protein 8 / Apoptotic protein NDPP1 / CARD-inhibitor of NF-kappa-B-activating ligand / CARDINAL / DACAR / Tumor ...Apoptotic protein NDPP1 / CARD-inhibitor of NF-kappa-B-activating ligand / CARDINAL / DACAR / Tumor up-regulated CARD-containing antagonist of CASP9 / TUCAN


Mass: 10103.373 Da / Num. of mol.: 16 / Fragment: CARD domain (UNP residues 451-537)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CARD8, KIAA0955, NDPP1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y2G2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: CARD8-CARD filament / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTrisC4H11NO31
2150 mMSodium ChlorideNaClSodium chloride1
31 mMTCEPC9H15O6P1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 magnification: 96000 X / Nominal defocus max: -2200 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.25 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1208

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionManually picked
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10REFMACmodel refinement
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -99.07 ° / Axial rise/subunit: 5.2 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 287568
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111333 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 4IKM

4ikm


Accession code: 4IKM / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00911136
ELECTRON MICROSCOPYf_angle_d0.79214912
ELECTRON MICROSCOPYf_dihedral_angle_d42.0861472
ELECTRON MICROSCOPYf_chiral_restr0.0471680
ELECTRON MICROSCOPYf_plane_restr0.0031984

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