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- PDB-7pzc: Cryo-EM structure of the NLRP3 decamer bound to the inhibitor CRID3 -

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Basic information

Entry
Database: PDB / ID: 7pzc
TitleCryo-EM structure of the NLRP3 decamer bound to the inhibitor CRID3
ComponentsNACHT, LRR and PYD domains-containing protein 3
KeywordsIMMUNE SYSTEM / NLRP3 / CP-456.773 / CRID3 / MCC950 / AAA+ ATPase / NOD-like receptor / inflammasome
Function / homology
Function and homology information


molecular sensor activity / detection of biotic stimulus / cysteine-type endopeptidase activator activity / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex / NLRP3 inflammasome complex assembly / interphase microtubule organizing center / positive regulation of T-helper 2 cell cytokine production / osmosensory signaling pathway ...molecular sensor activity / detection of biotic stimulus / cysteine-type endopeptidase activator activity / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex / NLRP3 inflammasome complex assembly / interphase microtubule organizing center / positive regulation of T-helper 2 cell cytokine production / osmosensory signaling pathway / positive regulation of type 2 immune response / pattern recognition receptor signaling pathway / peptidoglycan binding / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / microtubule organizing center / negative regulation of interleukin-1 beta production / pyroptotic inflammatory response / positive regulation of interleukin-4 production / negative regulation of acute inflammatory response / The NLRP3 inflammasome / protein maturation / Purinergic signaling in leishmaniasis infection / signaling adaptor activity / positive regulation of interleukin-1 beta production / molecular condensate scaffold activity / ADP binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein homooligomerization / Cytoprotection by HMOX1 / cellular response to virus / Metalloprotease DUBs / negative regulation of inflammatory response / defense response / positive regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / SARS-CoV-1 activates/modulates innate immune responses / positive regulation of NF-kappaB transcription factor activity / protein-macromolecule adaptor activity / cellular response to lipopolysaccharide / DNA-binding transcription factor binding / sequence-specific DNA binding / molecular adaptor activity / inflammatory response / Golgi membrane / innate immune response / apoptotic process / SARS-CoV-2 activates/modulates innate and adaptive immune responses / endoplasmic reticulum / signal transduction / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / mitochondrion / extracellular region / ATP binding / identical protein binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated domain / : / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / DAPIN domain / DAPIN domain profile. ...NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated domain / : / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat / Death-like domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-8GI / ADENOSINE-5'-DIPHOSPHATE / NACHT, LRR and PYD domains-containing protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHochheiser, I.V. / Pilsl, M. / Hagelueken, G. / Engel, C. / Geyer, M.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)EXC2151 - 390873048 Germany
CitationJournal: Nature / Year: 2022
Title: Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3.
Authors: Inga V Hochheiser / Michael Pilsl / Gregor Hagelueken / Jonas Moecking / Michael Marleaux / Rebecca Brinkschulte / Eicke Latz / Christoph Engel / Matthias Geyer /
Abstract: NLRP3 is an intracellular sensor protein that when activated by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation and pyroptosis. The conformational states of NLRP3 ...NLRP3 is an intracellular sensor protein that when activated by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation and pyroptosis. The conformational states of NLRP3 and the way antagonistic small molecules act at the molecular level remain poorly understood. Here we report the cryo-electron microscopy structures of full-length human NLRP3 in its native form and complexed with the inhibitor CRID3 (also named MCC950). Inactive, ADP-bound NLRP3 is a decamer composed of homodimers of intertwined leucine-rich repeat (LRR) domains that assemble back-to-back as pentamers. The NACHT domain is located at the apical axis of this spherical structure. One pyrin domain dimer is in addition formed inside the LRR cage. Molecular contacts between the concave sites of two opposing LRR domains are mediated by an acidic loop that extends from an LRR transition segment. Binding of CRID3 considerably stabilizes the NACHT and LRR domains relative to each other. CRID3 binds into a cleft, connecting four subdomains of the NACHT with the transition LRR. Its central sulfonylurea group interacts with the Walker A motif of the NLRP3 nucleotide-binding domain and is sandwiched between two arginine residues, which explains the specificity of NLRP3 for this chemical entity. With the determination of the binding site of this key therapeutic agent, specific targeting of NLRP3 for the treatment of autoinflammatory and autoimmune diseases and rational drug optimization is within reach.
History
DepositionOct 12, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: 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 / _citation_author.identifier_ORCID
Revision 1.2Apr 20, 2022Group: 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 1.3Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: NACHT, LRR and PYD domains-containing protein 3
B: NACHT, LRR and PYD domains-containing protein 3
C: NACHT, LRR and PYD domains-containing protein 3
D: NACHT, LRR and PYD domains-containing protein 3
E: NACHT, LRR and PYD domains-containing protein 3
F: NACHT, LRR and PYD domains-containing protein 3
G: NACHT, LRR and PYD domains-containing protein 3
H: NACHT, LRR and PYD domains-containing protein 3
I: NACHT, LRR and PYD domains-containing protein 3
J: NACHT, LRR and PYD domains-containing protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,191,55230
Polymers1,183,23510
Non-polymers8,31720
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, Multi-angle light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24030 Å2
ΔGint-152 kcal/mol
Surface area413570 Å2

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Components

#1: Protein
NACHT, LRR and PYD domains-containing protein 3 / Angiotensin/vasopressin receptor AII/AVP-like / Caterpiller protein 1.1 / CLR1.1 / Cold-induced ...Angiotensin/vasopressin receptor AII/AVP-like / Caterpiller protein 1.1 / CLR1.1 / Cold-induced autoinflammatory syndrome 1 protein / Cryopyrin / PYRIN-containing APAF1-like protein 1


Mass: 118323.531 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRP3, C1orf7, CIAS1, NALP3, PYPAF1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q96P20
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-8GI / 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(2-oxidanylpropan-2-yl)furan-2-yl]sulfonyl-urea


Mass: 404.480 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C20H24N2O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Decameric complex of the NLRP3 protein bound to ADP and CRID3
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.18 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 50 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 10 mM MgCl2, 1 mM ADP
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 defocus max: 1500 nm / Nominal defocus min: 750 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
10RELION3.1initial Euler assignment
11cryoSPARCv3.2initial Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1588061
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161351 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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