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- PDB-6x6a: Cryo-EM structure of NLRP1-DPP9 complex -

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Basic information

Entry
Database: PDB / ID: 6x6a
TitleCryo-EM structure of NLRP1-DPP9 complex
Components
  • (NACHT, LRR and PYD domains-containing protein 1) x 2
  • Dipeptidyl peptidase 9
KeywordsIMMUNE SYSTEM / NLRP1 / DPP9 / inflammasome / Val-boroPro (VbP) / talabostat / innate immunity
Function / homology
Function and homology information


NLRP1 inflammasome complex assembly / cysteine-type endopeptidase activator activity / NLRP1 inflammasome complex / canonical inflammasome complex / The NLRP1 inflammasome / dipeptidyl-peptidase IV / self proteolysis / dipeptidyl-peptidase activity / negative regulation of programmed cell death / Hydrolases; Acting on peptide bonds (peptidases) ...NLRP1 inflammasome complex assembly / cysteine-type endopeptidase activator activity / NLRP1 inflammasome complex / canonical inflammasome complex / The NLRP1 inflammasome / dipeptidyl-peptidase IV / self proteolysis / dipeptidyl-peptidase activity / negative regulation of programmed cell death / Hydrolases; Acting on peptide bonds (peptidases) / pattern recognition receptor activity / pattern recognition receptor signaling pathway / cellular response to UV-B / stress-activated protein kinase signaling cascade / pyroptosis / cell leading edge / antiviral innate immune response / cysteine-type endopeptidase activator activity involved in apoptotic process / response to muramyl dipeptide / signaling adaptor activity / aminopeptidase activity / molecular condensate scaffold activity / serine-type peptidase activity / positive regulation of interleukin-1 beta production / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein homooligomerization / positive regulation of inflammatory response / activation of cysteine-type endopeptidase activity involved in apoptotic process / : / double-stranded RNA binding / peptidase activity / regulation of inflammatory response / double-stranded DNA binding / defense response to virus / neuron apoptotic process / regulation of apoptotic process / microtubule / defense response to bacterium / protein domain specific binding / apoptotic process / nucleolus / enzyme binding / ATP hydrolysis activity / proteolysis / nucleoplasm / ATP binding / 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 / CARD8/ASC/NALP1, CARD domain / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain ...FIIND domain / Function to find / FIIND domain profile. / Dipeptidyl peptidase 8 /9 ,N-terminal / Dipeptidyl peptidase 8 and 9 N-terminal / CARD8/ASC/NALP1, CARD domain / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / Leucine rich repeat, ribonuclease inhibitor type / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / PAAD/DAPIN/Pyrin domain / Dipeptidylpeptidase IV, N-terminal domain / Dipeptidyl peptidase IV (DPP IV) N-terminal region / Leucine Rich repeat / Leucine Rich Repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Leucine-rich repeat profile. / Death-like domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Alpha/Beta hydrolase fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Dipeptidyl peptidase 9 / NACHT, LRR and PYD domains-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHollingsworth, L.R. / Sharif, H. / Griswold, A.R. / Fontana, P. / Mintseris, J. / Dagbay, K.B. / Paulo, J.A. / Gygi, S.P. / Bachovchin, D.A. / Wu, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 Al124491 United States
CitationJournal: Nature / Year: 2021
Title: DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation.
Authors: L Robert Hollingsworth / Humayun Sharif / Andrew R Griswold / Pietro Fontana / Julian Mintseris / Kevin B Dagbay / Joao A Paulo / Steven P Gygi / Daniel A Bachovchin / Hao Wu /
Abstract: Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and ...Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT). Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear. Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.
History
DepositionMay 27, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 10, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 31, 2021Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 12, 2021Group: 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 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Dipeptidyl peptidase 9
B: NACHT, LRR and PYD domains-containing protein 1
G: NACHT, LRR and PYD domains-containing protein 1
C: NACHT, LRR and PYD domains-containing protein 1
D: Dipeptidyl peptidase 9
E: NACHT, LRR and PYD domains-containing protein 1
I: NACHT, LRR and PYD domains-containing protein 1
F: NACHT, LRR and PYD domains-containing protein 1


Theoretical massNumber of molelcules
Total (without water)588,4668
Polymers588,4668
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dipeptidyl peptidase 9 / / DP9 / Dipeptidyl peptidase IV-related protein 2 / DPRP-2 / Dipeptidyl peptidase IX / DPP IX / ...DP9 / Dipeptidyl peptidase IV-related protein 2 / DPRP-2 / Dipeptidyl peptidase IX / DPP IX / Dipeptidyl peptidase-like protein 9 / DPLP9


Mass: 98384.320 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DPP9, DPRP2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q86TI2, dipeptidyl-peptidase IV
#2: Protein NACHT, LRR and PYD domains-containing protein 1 / Caspase recruitment domain-containing protein 7 / Death effector filament-forming ced-4-like ...Caspase recruitment domain-containing protein 7 / Death effector filament-forming ced-4-like apoptosis protein / Nucleotide-binding domain and caspase recruitment domain


Mass: 136327.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRP1, CARD7, DEFCAP, KIAA0926, NAC, NALP1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9C000
#3: Protein
NACHT, LRR and PYD domains-containing protein 1 / Caspase recruitment domain-containing protein 7 / Death effector filament-forming ced-4-like ...Caspase recruitment domain-containing protein 7 / Death effector filament-forming ced-4-like apoptosis protein / Nucleotide-binding domain and caspase recruitment domain


Mass: 29760.740 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRP1, CARD7, DEFCAP, KIAA0926, NAC, NALP1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9C000

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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: DPP9-NLRP1 complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: pcDNA3.1
Buffer solutionpH: 7.5 / Details: 25 mM Tris pH 7.5, 150 mM NaCl, 1 mM TCEP
SpecimenConc.: 0.5 mg/ml / Embedding 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: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: OTHER / Calibrated magnification: 10500 X / Nominal defocus max: 2500 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

IDAverage exposure time (sec.)Electron dose (e/Å2)Num. of grids imagedNum. of real imagesDetails
11.867.5477840stage tilt 0 degrees
22.667.641916stage tilt 37 degrees

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.7image acquisition
4CTFFIND9.03CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11197166
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179306 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01520870
ELECTRON MICROSCOPYf_angle_d1.82428339
ELECTRON MICROSCOPYf_dihedral_angle_d12.1082740
ELECTRON MICROSCOPYf_chiral_restr0.1093015
ELECTRON MICROSCOPYf_plane_restr0.0133659

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