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- EMDB-22974: Structure of DPP9 bound to catalytically-inactive CARD8 (S297A) -

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Basic information

Entry
Database: EMDB / ID: EMD-22974
TitleStructure of DPP9 bound to catalytically-inactive CARD8 (S297A)
Map data
Sample
  • Cell: DPP9 bound to catalytically-inactive CARD8 (S297A)
    • Other: DPP9
    • Other: CARD8
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 / dipeptidyl-peptidase IV / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway ...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 / dipeptidyl-peptidase IV / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway / self proteolysis / dipeptidyl-peptidase activity / Regulation of the apoptosome activity / negative regulation of programmed cell death / 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 / pyroptotic inflammatory response / cell leading edge / cysteine-type endopeptidase activator activity involved in apoptotic process / antiviral innate immune response / 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 / aminopeptidase activity / serine-type peptidase activity / molecular condensate scaffold activity / positive regulation of interleukin-1 beta production / peptidase activity / regulation of apoptotic process / defense response to virus / microtubule / protein homodimerization activity / protein-containing complex / proteolysis / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
FIIND domain / Function to find / FIIND domain profile. / Dipeptidyl peptidase 8 /9 ,N-terminal / Dipeptidyl peptidase 8 and 9 N-terminal / Dipeptidylpeptidase IV, N-terminal domain / Dipeptidyl peptidase IV (DPP IV) N-terminal region / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain ...FIIND domain / Function to find / FIIND domain profile. / Dipeptidyl peptidase 8 /9 ,N-terminal / Dipeptidyl peptidase 8 and 9 N-terminal / Dipeptidylpeptidase IV, N-terminal domain / Dipeptidyl peptidase IV (DPP IV) N-terminal region / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Death-like domain superfamily / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Dipeptidyl peptidase 9 / Caspase recruitment domain-containing protein 8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsHollingsworth LR / Sharif H / Wu H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5DP1HD087988-02 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5R01AI124491-03 United States
CitationJournal: Immunity / Year: 2021
Title: Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.
Authors: Humayun Sharif / L Robert Hollingsworth / Andrew R Griswold / Jeffrey C Hsiao / Qinghui Wang / Daniel A Bachovchin / Hao Wu /
Abstract: CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) ...CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability.
History
DepositionNov 9, 2020-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0639
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0639
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22974.png

Downloads & links

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Map

FileDownload / File: emd_22974.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0639 / Movie #1: 0.0639
Minimum - Maximum-0.10709115 - 0.24159396
Average (Standard dev.)0.0005617407 (±0.01044621)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z265.600265.600265.600
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1070.2420.001

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Supplemental data

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Additional map: Sharpened Map

Fileemd_22974_additional_1.map
AnnotationSharpened Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DPP9 bound to catalytically-inactive CARD8 (S297A)

EntireName: DPP9 bound to catalytically-inactive CARD8 (S297A)
Components
  • Cell: DPP9 bound to catalytically-inactive CARD8 (S297A)
    • Other: DPP9
    • Other: CARD8

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Supramolecule #1: DPP9 bound to catalytically-inactive CARD8 (S297A)

SupramoleculeName: DPP9 bound to catalytically-inactive CARD8 (S297A) / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DPP9

MacromoleculeName: DPP9 / type: other / ID: 1 / Classification: other
Source (natural)Organism: Homo sapiens (human)
SequenceString: GAMGSMATTG TPTADRGDAA ATDDPAARFQ VQKHSWDGLR SIIHGSRKYS GLIVNKAPHD FQFVQKTDES GPHSHRLYYL GMPYGSRENS LLYSEIPKKV RKEALLLLSW KQMLDHFQAT PHHGVYSREE ELLRERKRLG VFGITSYDFH SESGLFLFQA SNSLFHCRDG ...String:
GAMGSMATTG TPTADRGDAA ATDDPAARFQ VQKHSWDGLR SIIHGSRKYS GLIVNKAPHD FQFVQKTDES GPHSHRLYYL GMPYGSRENS LLYSEIPKKV RKEALLLLSW KQMLDHFQAT PHHGVYSREE ELLRERKRLG VFGITSYDFH SESGLFLFQA SNSLFHCRDG GKNGFMVSPM KPLEIKTQCS GPRMDPKICP ADPAFFSFIN NSDLWVANIE TGEERRLTFC HQGLSNVLDD PKSAGVATFV IQEEFDRFTG YWWCPTASWE GSEGLKTLRI LYEEVDESEV EVIHVPSPAL EERKTDSYRY PRTGSKNPKI ALKLAEFQTD SQGKIVSTQE KELVQPFSSL FPKVEYIARA GWTRDGKYAW AMFLDRPQQW LQLVLLPPAL FIPSTENEEQ RLASARAVPR NVQPYVVYEE VTNVWINVHD IFYPFPQSEG EDELCFLRAN ECKTGFCHLY KVTAVLKSQG YDWSEPFSPG EDEFKCPIKE EIALTSGEWE VLARHGSKIW VNEETKLVYF QGTKDTPLEH HLYVVSYEAA GEIVRLTTPG FSHSCSMSQN FDMFVSHYSS VSTPPCVHVY KLSGPDDDPL HKQPRFWASM MEAASCPPDY VPPEIFHFHT RSDVRLYGMI YKPHALQPGK KHPTVLFVYG GPQVQLVNNS FKGIKYLRLN TLASLGYAVV VIDGRGSCQR GLRFEGALKN QMGQVEIEDQ VEGLQFVAEK YGFIDLSRVA IHGWSYGGFL SLMGLIHKPQ VFKVAIAGAP VTVWMAYDTG YTERYMDVPE NNQHGYEAGS VALHVEKLPN EPNRLLILHG FLDENVHFFH TNFLVSQLIR AGKPYQLQIY PNERHSIRCP ESGEHYEVTL LHFLQEYL
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #2: CARD8

MacromoleculeName: CARD8 / type: other / ID: 2 / Classification: other
Source (natural)Organism: Homo sapiens (human)
SequenceString: MEKKECPEKS SSSEEELPRR DSGSSRNIDA SKLIRLQGSR KLLVDNSIRE LQYTKTGIFF QAEACVTNDT VYRELPCVSE TLCDISHFFQ EDDETEAEPL LFRAVPECQL SGGDIPSVSE EQESSEGQDS GDICSEENQI VSSYASKVCF EIEEDYKNRQ FLGPEGNVDV ...String:
MEKKECPEKS SSSEEELPRR DSGSSRNIDA SKLIRLQGSR KLLVDNSIRE LQYTKTGIFF QAEACVTNDT VYRELPCVSE TLCDISHFFQ EDDETEAEPL LFRAVPECQL SGGDIPSVSE EQESSEGQDS GDICSEENQI VSSYASKVCF EIEEDYKNRQ FLGPEGNVDV ELIDKSTNRY SVWFPTAGWY LWSATGLGFL VRDEVTVTIA FGSWSQHLAL DLQHHEQWLV GGPLFDVTAE PEEAVAEIHL PHFISLQAGE VDVSWFLVAH FKNEGMVLEH PARVEPFYAV LESPSFALMG ILLRIASGTR LSIPITSNTL IYYHPHPEDI KFHLYLVPSD ALLTKAIDDE EDRFHGVRLQ TSPPMEPLNF GSSYIVSNSA NLKVMPKELK LSYRSPGEIQ HFSKFYAGQM KEPIQLEITE KRHGTLVWDT EVKPVDLQLV AASAPPPFSG AAFVKENHRQ LQARMGDLKG VLDDLQDNEV LTENEKELVE QEKTRQSKNE ALLSMVEKKG DLALDVLFRS ISERDPYLVS YLRQQNL
Recombinant expressionOrganism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.40 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
25.0 mMC8H18N2O4SHEPES
1.0 mMC9H15O6PTCEP
0.02 %C5H8O2Glutaraldehyde

Details: Crosslinking with glutaraldehyde was done for 5-10 minutes on ice immediately before plunging grids.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -0.0025 µm / Nominal defocus min: -0.0008 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 3840 / Average exposure time: 1.5 sec. / Average electron dose: 63.67 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1634615
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15.0)
Startup modelType of model: OTHER / Details: Ab-initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15.0)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 2.15.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0) / Number images used: 92404
FSC plot (resolution estimation)

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