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- EMDB-73688: Cryo-EM structure of VVD-908 NLRP3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-73688
TitleCryo-EM structure of VVD-908 NLRP3 complex
Map dataSharpened map after symmetry expansion and focused refinement on residues 135-675
Sample
  • Complex: NLRP3 hexamer
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: (4S,5S)-1-[(2E)-but-2-enoyl]-7-(3-chloro-5-fluorophenyl)-4-phenyl-1,7-diazaspiro[4.4]nonan-6-one
  • Ligand: MAGNESIUM ION
Keywordsinflammasome / NLRP3 / allosteric / IMMUNE SYSTEM
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
NACHT, LRR and PYD domains-containing protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsBernard SM
Funding support1 items
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, 2025-
Header (metadata) releaseAug 19, 2026-
Map releaseAug 19, 2026-
UpdateAug 19, 2026-
Current statusAug 19, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73688.png

Downloads & links

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Map

FileDownload / File: emd_73688.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map after symmetry expansion and focused refinement on residues 135-675
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.165
Minimum - Maximum-1.5636 - 2.1465304
Average (Standard dev.)0.003100494 (±0.029892059)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Additional map: Map after symmetry expansion and focused refinement on...

Fileemd_73688_additional_1.map
AnnotationMap after symmetry expansion and focused refinement on residues 135-675
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_73688_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_73688_half_map_2.map
AnnotationHalf map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : NLRP3 hexamer

EntireName: NLRP3 hexamer
Components
  • Complex: NLRP3 hexamer
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: (4S,5S)-1-[(2E)-but-2-enoyl]-7-(3-chloro-5-fluorophenyl)-4-phenyl-1,7-diazaspiro[4.4]nonan-6-one
  • Ligand: MAGNESIUM ION

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Supramolecule #1: NLRP3 hexamer

SupramoleculeName: NLRP3 hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 615 KDa

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Macromolecule #1: NACHT, LRR and PYD domains-containing protein 3

MacromoleculeName: NACHT, LRR and PYD domains-containing protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 102.690508 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DYRKKYRKYV RSRFQCIEDR NARLGESVSL NKRYTRLRLI KEHRSQQERE QELLAIGKTK TCESPVSPIK MELLFDPDDE HSEPVHTVV FQGAAGIGKT ILARKMMLDW ASGTLYQDRF DYLFYIHCRE VSLVTQRSLG DLIMSCCPDP NPPIHKIVRK P SRILFLMD ...String:
DYRKKYRKYV RSRFQCIEDR NARLGESVSL NKRYTRLRLI KEHRSQQERE QELLAIGKTK TCESPVSPIK MELLFDPDDE HSEPVHTVV FQGAAGIGKT ILARKMMLDW ASGTLYQDRF DYLFYIHCRE VSLVTQRSLG DLIMSCCPDP NPPIHKIVRK P SRILFLMD GFDELQGAFD EHIGPLCTDW QKAERGDILL SSLIRKKLLP EASLLITTRP VALEKLQHLL DHPRHVEILG FS EAKRKEY FFKYFSDEAQ ARAAFSLIQE NEVLFTMCFI PLVCWIVCTG LKQQMESGKS LAQTSKTTTA VYVFFLSSLL QPR GGSQEH GLCAHLWGLC SLAADGIWNQ KILFEESDLR NHGLQKADVS AFLRMNLFQK EVDCEKFYSF IHMTFQEFFA AMYY LLEEE KEGRTNVPGS RLKLPSRDVT VLLENYGKFE KGYLIFVVRF LFGLVNQERT SYLEKKLSCK ISQQIRLELL KWIEV KAKA KKLQIQPSQL ELFYCLYEMQ EEDFVQRAMD YFPKIEINLS TRMDHMVSSF CIENCHRVES LSLGFLHNMP KEEEEE EKE GRHLDMVQCV LPSSSHAACS HGLVNSHLTS SFCRGLFSVL STSQSLTELD LSDNSLGDPG MRVLCETLQH PGCNIRR LW LGRCGLSHEC CFDISLVLSS NQKLVELDLS DNALGDFGIR LLCVGLKHLL CNLKKLWLVS CCLTSACCQD LASVLSTS H SLTRLYVGEN ALGDSGVAIL CEKAKNPQCN LQKLGLVNSG LTSVCCSALS SVLSTNQNLT HLYLRGNTLG DKGIKLLCE GLLHPDCKLQ VLELDNCNLT SHCCWDLSTL LTSSQSLRKL SLGNNDLGDL GVMMFCEVLK QQSCLLQNLG LSEMYFNYET KSALETLQE EKPELTVVFE PSW

UniProtKB: NACHT, LRR and PYD domains-containing protein 3

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: (4S,5S)-1-[(2E)-but-2-enoyl]-7-(3-chloro-5-fluorophenyl)-4-phenyl...

MacromoleculeName: (4S,5S)-1-[(2E)-but-2-enoyl]-7-(3-chloro-5-fluorophenyl)-4-phenyl-1,7-diazaspiro[4.4]nonan-6-one
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1CZP
Molecular weightTheoretical: 412.884 Da

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7vtp

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 870000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION

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