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- PDB-9z03: Cryo-EM structure of VVD-908 NLRP3 complex -

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Basic information

Entry
Database: PDB / ID: 9z03
TitleCryo-EM structure of VVD-908 NLRP3 complex
ComponentsNACHT, LRR and PYD domains-containing protein 3
KeywordsIMMUNE SYSTEM / inflammasome / NLRP3 / allosteric
Function / homology
Function and homology information


detection of biotic stimulus / molecular sensor activity / positive regulation of type 2 immune response / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / positive regulation of T-helper 2 cell cytokine production / interphase microtubule organizing center / NLRP3 inflammasome complex / peptidoglycan binding / NLRP3 inflammasome complex assembly ...detection of biotic stimulus / molecular sensor activity / positive regulation of type 2 immune response / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / positive regulation of T-helper 2 cell cytokine production / interphase microtubule organizing center / NLRP3 inflammasome complex / peptidoglycan binding / NLRP3 inflammasome complex assembly / cysteine-type endopeptidase activator activity / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / osmosensory signaling pathway / negative regulation of interleukin-1 beta production / pattern recognition receptor signaling pathway / positive regulation of interleukin-4 production / negative regulation of acute inflammatory response / microtubule organizing center / The NLRP3 inflammasome / pyroptotic inflammatory response / Purinergic signaling in leishmaniasis infection / signaling adaptor activity / positive regulation of interleukin-1 beta production / cellular response to virus / protein maturation / defense response / molecular condensate scaffold activity / Cytoprotection by HMOX1 / negative regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / protein homooligomerization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / positive regulation of inflammatory response / Metalloprotease DUBs / SARS-CoV-1 activates/modulates innate immune responses / cellular response to lipopolysaccharide / regulation of inflammatory response / DNA-binding transcription factor binding / sequence-specific DNA binding / molecular adaptor activity / protein-macromolecule adaptor activity / inflammatory response / Golgi membrane / innate immune response / apoptotic process / SARS-CoV-2 activates/modulates innate and adaptive immune responses / signal transduction / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / mitochondrion / DNA-templated transcription / extracellular region / ATP binding / membrane / identical protein binding / 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 profile. / NACHT nucleoside triphosphatase ...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 profile. / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / DAPIN domain / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / 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
: / 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: 3.03 Å
AuthorsBernard, S.M.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Br J Pharmacol / Year: 2026
Title: Chemoproteomic discovery of a brain-penetrant, covalent NLRP3 inhibitor that binds a novel allosteric pocket.
Authors: Donald C Rogness / Evelyn P Sievert / Vincent F Vartabedian / Steffen M Bernard / Erick Aitchison / William Tao / Mikaela Lindvall / Jing Qian / Christie L Eissler / Brian E Nordin / ...Authors: Donald C Rogness / Evelyn P Sievert / Vincent F Vartabedian / Steffen M Bernard / Erick Aitchison / William Tao / Mikaela Lindvall / Jing Qian / Christie L Eissler / Brian E Nordin / Benjamin D Horning / Cian Kingston / Kelsey N Lamb / Joshua C Bell / Bingwen Lu / Jonathan Pollock / Jun Shi / Roli Khattri / David S Weinstein / Matthew P Patricelli / Brian N Cook / Gabriel M Simon /
Abstract: BACKGROUND AND PURPOSE: The NLRP3 inflammasome is an attractive therapeutic target for multiple inflammatory conditions. Although inhibitors have been developed, their chemical diversity is limited, ...BACKGROUND AND PURPOSE: The NLRP3 inflammasome is an attractive therapeutic target for multiple inflammatory conditions. Although inhibitors have been developed, their chemical diversity is limited, and their properties are not ideal for brain penetrance, which is desirable for treating neuroinflammatory disorders.
EXPERIMENTAL APPROACH: We applied our chemoproteomics platform to survey our electrophilic fragment collection to identify inhibitors of NLRP3. We focused our attention on compounds that bind Cys463, ...EXPERIMENTAL APPROACH: We applied our chemoproteomics platform to survey our electrophilic fragment collection to identify inhibitors of NLRP3. We focused our attention on compounds that bind Cys463, as this residue was identified as an allosteric sensor of NLRP3 function.
KEY RESULTS: A novel inhibitor series was identified bearing a butynamide electrophile and a unique spirocyclic lactam core. Compounds from this series displayed mid-nanomolar potency and were found ...KEY RESULTS: A novel inhibitor series was identified bearing a butynamide electrophile and a unique spirocyclic lactam core. Compounds from this series displayed mid-nanomolar potency and were found to inhibit IL-1β secretion in a Cys463-dependent manner. Cryo-EM structures revealed that ligand binding to Cys463 stabilizes an inactive conformation, thereby preventing structural rearrangements required for inflammasome activation. These compounds displayed attractive pharmacokinetic properties and, notably, Kp,uu values >0.5, suggesting the potential to address neuroinflammatory disorders. Administration of a representative compound to humanized mice resulted in clear NLRP3 Cys463 target-engagement and profound suppression of LPS- and ATP-induced IL-1β secretion, demonstrating clear proof-of-concept in vivo.
CONCLUSION AND IMPLICATIONS: Chemoproteomics-based ligand discovery is intrinsically function-agnostic and has the potential to identify novel pockets on even well-characterized protein targets. ...CONCLUSION AND IMPLICATIONS: Chemoproteomics-based ligand discovery is intrinsically function-agnostic and has the potential to identify novel pockets on even well-characterized protein targets. Here, optimization of ligands targeting Cys463 of NLRP3 within a previously uncharacterized allosteric pocket led to a unique and potent inhibitor series with attractive physicochemical and pharmacokinetic properties for the potential treatment of diseases involving aberrant innate immune activation in both central and peripheral tissues.
History
DepositionOct 30, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NACHT, LRR and PYD domains-containing protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,5554
Polymers102,6911
Non-polymers8643
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 102690.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRP3, C1orf7, CIAS1, NALP3, PYPAF1 / Production host: Homo sapiens (human)
References: UniProt: Q96P20, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-A1CZP / (4S,5S)-1-[(2E)-but-2-enoyl]-7-(3-chloro-5-fluorophenyl)-4-phenyl-1,7-diazaspiro[4.4]nonan-6-one


Mass: 412.884 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H22ClFN2O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY
Has protein modificationY

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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: NLRP3 hexamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.615 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS 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: 800 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1Topazparticle selection
2PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 870000 / Symmetry type: POINT
RefinementHighest resolution: 3.03 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056496
ELECTRON MICROSCOPYf_angle_d0.4988767
ELECTRON MICROSCOPYf_dihedral_angle_d6.783863
ELECTRON MICROSCOPYf_chiral_restr0.04998
ELECTRON MICROSCOPYf_plane_restr0.0031093

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