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- PDB-8fvu: Cryo-EM structure of human Needle/NAIP/NLRC4 (R288A) -

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Basic information

Entry
Database: PDB / ID: 8fvu
TitleCryo-EM structure of human Needle/NAIP/NLRC4 (R288A)
Components
  • Baculoviral IAP repeat-containing protein 1
  • Lethal factor,Type III secretion system protein
  • NLR family CARD domain-containing protein 4
KeywordsIMMUNE SYSTEM / NLRC4 / NAIP / Inflammasome
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / IPAF inflammasome complex / The IPAF inflammasome / positive regulation of protein processing / icosanoid biosynthetic process / type III protein secretion system complex / caspase binding / protein secretion by the type III secretion system / canonical inflammasome complex / cysteine-type endopeptidase inhibitor activity involved in apoptotic process ...anthrax lethal factor endopeptidase / IPAF inflammasome complex / The IPAF inflammasome / positive regulation of protein processing / icosanoid biosynthetic process / type III protein secretion system complex / caspase binding / protein secretion by the type III secretion system / canonical inflammasome complex / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / host cell cytosol / pattern recognition receptor signaling pathway / TP53 Regulates Transcription of Caspase Activators and Caspases / Uptake and function of anthrax toxins / pyroptotic inflammatory response / detection of bacterium / negative regulation of tumor necrosis factor-mediated signaling pathway / cysteine-type endopeptidase inhibitor activity / endopeptidase activator activity / activation of innate immune response / positive regulation of interleukin-1 beta production / toxin activity / protein serine/threonine kinase binding / metalloendopeptidase activity / positive regulation of JNK cascade / protein homooligomerization / metallopeptidase activity / positive regulation of inflammatory response / nervous system development / negative regulation of neuron apoptotic process / innate immune response / basolateral plasma membrane / regulation of apoptotic process / defense response to bacterium / inflammatory response / apoptotic process / positive regulation of apoptotic process / negative regulation of apoptotic process / magnesium ion binding / cell surface / protein homodimerization activity / proteolysis / metal ion binding / zinc ion binding / ATP binding / extracellular region / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / : / NLRC4 LRR domain / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain ...Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / : / NLRC4 LRR domain / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily / Type III secretion needle MxiH, YscF, SsaG, EprI, PscF, EscF / Type III secretion system, needle protein / Anthrax toxin, lethal/endema factor / Anthrax toxin, lethal/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain / Anthrax toxin lethal factor (ATLF)-like domain profile. / BIR repeat. / NACHT domain / NACHT nucleoside triphosphatase / NACHT-NTPase domain profile. / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Metallopeptidase, catalytic domain superfamily / Death-like domain superfamily / Leucine-rich repeat domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Lethal factor / Baculoviral IAP repeat-containing protein 1 / Type III secretion system protein / NLR family CARD domain-containing protein 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Bacillus anthracis (anthrax bacterium)
Burkholderia (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMatico, R.E. / Yu, X. / Miller, R. / Somani, S. / Ricketts, M.D. / Kumar, N. / Steele, R.A. / Medley, Q. / Berger, S. / Faustin, B. / Sharma, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.
Authors: Rosalie E Matico / Xiaodi Yu / Robyn Miller / Sandeep Somani / M Daniel Ricketts / Nikit Kumar / Ruth A Steele / Quintus Medley / Scott Berger / Benjamin Faustin / Sujata Sharma /
Abstract: The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in ...The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.
History
DepositionJan 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jan 31, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Baculoviral IAP repeat-containing protein 1
B: NLR family CARD domain-containing protein 4
U: Lethal factor,Type III secretion system protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)320,9607
Polymers320,2573
Non-polymers7034
Water00
1


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

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Components

#1: Protein Baculoviral IAP repeat-containing protein 1 / Neuronal apoptosis inhibitory protein


Mass: 160484.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NAIP, BIRC1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13075
#2: Protein NLR family CARD domain-containing protein 4 / CARD / LRR / and NACHT-containing protein / CED-4-like protein Clan / Caspase recruitment domain- ...CARD / LRR / and NACHT-containing protein / CED-4-like protein Clan / Caspase recruitment domain-containing protein 12 / Ice protease-activating factor / Ipaf


Mass: 116941.531 Da / Num. of mol.: 1 / Mutation: R288A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRC4, CARD12, CLAN, CLAN1, IPAF, UNQ6189/PRO20215 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NPP4
#3: Protein Lethal factor,Type III secretion system protein / LF / Anthrax lethal toxin endopeptidase component


Mass: 42830.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus anthracis (anthrax bacterium), (gene. exp.) Burkholderia (bacteria)
Gene: lef, pXO1-107, BXA0172, GBAA_pXO1_0172, BPSS1548 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P15917, UniProt: Q63K18, anthrax lethal factor endopeptidase
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / 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

Component
IDNameTypeEntity IDParent-IDSource
1the complex of Needle, human NAIP and NLRC4COMPLEX#1-#30MULTIPLE SOURCES
2human NAIP and NLRC4COMPLEX#1-#21RECOMBINANT
3Burkholderia Needle (chimeric with an anthrax toxin lethal factor tag)COMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Bacillus anthracis (anthrax bacterium)1392
43Burkholderia (bacteria)32008
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Spodoptera frugiperda (fall armyworm)7108
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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106381 / Symmetry type: POINT

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