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- PDB-7keu: Cryo-EM structure of the Caspase-1-CARD:ASC-CARD octamer -

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Basic information

Entry
Database: PDB / ID: 7keu
TitleCryo-EM structure of the Caspase-1-CARD:ASC-CARD octamer
Components
  • Apoptosis-associated speck-like protein containing a CARD
  • Caspase-1
KeywordsIMMUNE SYSTEM / ASC / Apoptosis-associated speck-like protein containing a CARD / PYCARD / caspase-1 / cryo-EM / helical filament / octamer
Function / homology
Function and homology information


Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / caspase-1 / protease inhibitor complex / myeloid dendritic cell activation / IkappaB kinase complex ...Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / caspase-1 / protease inhibitor complex / myeloid dendritic cell activation / IkappaB kinase complex / AIM2 inflammasome complex assembly / The AIM2 inflammasome / AIM2 inflammasome complex / IPAF inflammasome complex / macropinocytosis / The IPAF inflammasome / icosanoid biosynthetic process / NLRP1 inflammasome complex / interleukin-6 receptor binding / cytokine precursor processing / canonical inflammasome complex / positive regulation of interleukin-18 production / NLRP3 inflammasome complex assembly / BMP receptor binding / NLRP3 inflammasome complex / positive regulation of adaptive immune response / caspase binding / CARD domain binding / osmosensory signaling pathway / negative regulation of protein serine/threonine kinase activity / negative regulation of interferon-beta production / CLEC7A/inflammasome pathway / positive regulation of cysteine-type endopeptidase activity / positive regulation of tumor necrosis factor-mediated signaling pathway / Interleukin-1 processing / regulation of tumor necrosis factor-mediated signaling pathway / Interleukin-37 signaling / positive regulation of extrinsic apoptotic signaling pathway / pattern recognition receptor signaling pathway / positive regulation of macrophage cytokine production / negative regulation of NF-kappaB transcription factor activity / positive regulation of actin filament polymerization / tropomyosin binding / signaling receptor ligand precursor processing / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response / : / positive regulation of activated T cell proliferation / positive regulation of release of cytochrome c from mitochondria / cytokine binding / cysteine-type endopeptidase activator activity involved in apoptotic process / intrinsic apoptotic signaling pathway by p53 class mediator / positive regulation of interleukin-10 production / The NLRP3 inflammasome / protein autoprocessing / protein maturation / cellular response to interleukin-1 / negative regulation of cytokine production involved in inflammatory response / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / positive regulation of T cell migration / Pyroptosis / Purinergic signaling in leishmaniasis infection / negative regulation of canonical NF-kappaB signal transduction / positive regulation of phagocytosis / positive regulation of chemokine production / positive regulation of defense response to virus by host / tumor necrosis factor-mediated signaling pathway / activation of innate immune response / positive regulation of interleukin-1 beta production / regulation of autophagy / positive regulation of interleukin-8 production / positive regulation of JNK cascade / positive regulation of DNA-binding transcription factor activity / regulation of protein stability / NOD1/2 Signaling Pathway / protein homooligomerization / : / kinase binding / cellular response to type II interferon / positive regulation of inflammatory response / positive regulation of interleukin-6 production / cellular response to mechanical stimulus / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of type II interferon production / azurophil granule lumen / SARS-CoV-1 activates/modulates innate immune responses / positive regulation of tumor necrosis factor production / positive regulation of T cell activation / cellular response to tumor necrosis factor / positive regulation of NF-kappaB transcription factor activity / regulation of inflammatory response / cellular response to lipopolysaccharide / protease binding / secretory granule lumen / defense response to virus / regulation of apoptotic process / defense response to Gram-negative bacterium / positive regulation of canonical NF-kappaB signal transduction / endopeptidase activity / transmembrane transporter binding
Similarity search - Function
CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / Caspase recruitment domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain ...CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / Caspase recruitment domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment 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
Caspase-1 / Apoptosis-associated speck-like protein containing a CARD
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHollingsworth, L.R. / David, L. / Li, Y. / Ruan, J. / 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
DepositionOct 12, 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 / struct_ncs_dom_lim
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 / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

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Assembly

Deposited unit
E: Caspase-1
F: Caspase-1
G: Caspase-1
H: Caspase-1
A: Apoptosis-associated speck-like protein containing a CARD
B: Apoptosis-associated speck-like protein containing a CARD
C: Apoptosis-associated speck-like protein containing a CARD
D: Apoptosis-associated speck-like protein containing a CARD


Theoretical massNumber of molelcules
Total (without water)77,0098
Polymers77,0098
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11E
21F
12E
22G
13E
23H
14F
24G
15F
25H
16A
26B
17A
27C
18A
28D
19B
29C
110B
210D
111C
211D

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ALAALATHRTHREA2 - 861 - 85
21ALAALATHRTHRFB2 - 861 - 85
12ALAALATHRTHREA2 - 861 - 85
22ALAALATHRTHRGC2 - 861 - 85
13ALAALATHRTHREA2 - 861 - 85
23ALAALATHRTHRHD2 - 861 - 85
14ALAALATHRTHRFB2 - 861 - 85
24ALAALATHRTHRGC2 - 861 - 85
15ALAALATHRTHRFB2 - 861 - 85
25ALAALATHRTHRHD2 - 861 - 85
16HISHISARGARGAE113 - 1941 - 82
26HISHISARGARGBF113 - 1941 - 82
17HISHISARGARGAE113 - 1941 - 82
27HISHISARGARGCG113 - 1941 - 82
18HISHISARGARGAE113 - 1941 - 82
28HISHISARGARGDH113 - 1941 - 82
19HISHISARGARGBF113 - 1941 - 82
29HISHISARGARGCG113 - 1941 - 82
110HISHISARGARGBF113 - 1941 - 82
210HISHISARGARGDH113 - 1941 - 82
111HISHISARGARGCG113 - 1941 - 82
211HISHISARGARGDH113 - 1941 - 82

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11

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Components

#1: Protein
Caspase-1 / CASP-1 / Interleukin-1 beta convertase / IL-1BC / Interleukin-1 beta-converting enzyme / IL-1 beta- ...CASP-1 / Interleukin-1 beta convertase / IL-1BC / Interleukin-1 beta-converting enzyme / IL-1 beta-converting enzyme / p45


Mass: 9719.329 Da / Num. of mol.: 4 / Fragment: UNP residues 2-86
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CASP1, IL1BC, IL1BCE / Production host: Escherichia coli (E. coli) / References: UniProt: P29466, caspase-1
#2: Protein
Apoptosis-associated speck-like protein containing a CARD / hASC / Caspase recruitment domain-containing protein 5 / PYD and CARD domain-containing protein / ...hASC / Caspase recruitment domain-containing protein 5 / PYD and CARD domain-containing protein / Target of methylation-induced silencing 1


Mass: 9532.825 Da / Num. of mol.: 4 / Fragment: UNP residues 113-194
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PYCARD, ASC, CARD5, TMS1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9ULZ3

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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-1-CARD:ASC-CARD octamer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.0863 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClC4H11NO31
2150 mMSodium ChlorideNaCl1
32 mMDTTC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.12 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5377
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: REFMAC / Version: 5.8.0258 / Classification: refinement
EM software
IDNameVersionCategory
2Gautomatchparticle selection
8PHENIXmodel fitting
9Cootmodel fitting
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
15PHENIXmodel refinement
16REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1863457
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111053 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15FNA

5fna

15FNA1PDBexperimental model
26N1H

6n1h

16N1H2PDBexperimental model
RefinementResolution: 3.9→198 Å / Cor.coef. Fo:Fc: 0.831 / SU B: 23.967 / SU ML: 0.344 / ESU R: 0.252
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.4451 --
obs0.4451 274049 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 105.715 Å2
Baniso -1Baniso -2Baniso -3
1--4.34 Å2-0.3 Å21.08 Å2
2---2.77 Å2-0.81 Å2
3---7.11 Å2
Refinement stepCycle: 1 / Total: 5306
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0125382
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.6371.647270
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.7285660
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.44822.526289
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.58515997
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.9111540
ELECTRON MICROSCOPYr_chiral_restr0.1710.2716
ELECTRON MICROSCOPYr_gen_planes_refined0.010.024028
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it5.68910.0042664
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it9.4215.0093316
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it6.24411.2782718
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined21.01622709
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11E40340.26
12F40340.26
21E41480.27
22G41480.27
31E40360.3
32H40360.3
41F41860.26
42G41860.26
51F41500.29
52H41500.29
61A42600.24
62B42600.24
71A42780.23
72C42780.23
81A43340.21
82D43340.21
91B43020.25
92C43020.25
101B43440.22
102D43440.22
111C44240.23
112D44240.23
LS refinement shellResolution: 3.9→4.002 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.492 20482 -
obs--100 %

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