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- EMDB-24387: Cryo-EM structure of the unliganded form of NLR family apoptosis ... -

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Basic information

Entry
Database: EMDB / ID: EMD-24387
TitleCryo-EM structure of the unliganded form of NLR family apoptosis inhibitory protein 5 (NAIP5)
Map data
Sample
  • Cell: pre-liganded form of NAIP5
    • Protein or peptide: Baculoviral IAP repeat-containing protein 1e
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Function / homology
Function and homology information


IPAF inflammasome complex / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / positive regulation of interleukin-1 beta production / perikaryon / defense response to Gram-negative bacterium / defense response to bacterium / inflammatory response / symbiont entry into host cell ...IPAF inflammasome complex / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / positive regulation of interleukin-1 beta production / perikaryon / defense response to Gram-negative bacterium / defense response to bacterium / inflammatory response / symbiont entry into host cell / innate immune response / neuronal cell body / negative regulation of apoptotic process / apoptotic process / ATP binding / metal ion binding
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / BIR repeat. / BIR repeat ...Baculoviral IAP repeat-containing protein 1 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / BIR repeat. / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Baculoviral IAP repeat-containing protein 1e
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsPaidimuddala B / Cao J / Xie Q / Wu H / Zhang L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanism of NAIP-NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5.
Authors: Bhaskar Paidimuddala / Jianhao Cao / Grady Nash / Qing Xie / Hao Wu / Liman Zhang /
Abstract: The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in ...The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP-NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17-18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5-NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation.
History
DepositionJul 2, 2021-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24387.png

Downloads & links

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Map

FileDownload / File: emd_24387.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.766 Å		1.07 Å/pix.
x 256 pix.
= 273.766 Å		1.07 Å/pix.
x 256 pix.
= 273.766 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0694 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.0016931666 - 1.9440949
Average (Standard dev.)0.0009218555 (±0.023368588)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.7664 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_24387_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_24387_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : pre-liganded form of NAIP5

EntireName: pre-liganded form of NAIP5
Components
  • Cell: pre-liganded form of NAIP5
    • Protein or peptide: Baculoviral IAP repeat-containing protein 1e
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: pre-liganded form of NAIP5

SupramoleculeName: pre-liganded form of NAIP5 / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Baculoviral IAP repeat-containing protein 1e

MacromoleculeName: Baculoviral IAP repeat-containing protein 1e / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 161.128156 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKL AEHGESSEDR ISEIDYEFLP ELSALLGVDA FQVAKSQEEE EHKERMKMKK GFNSQMRSEA KRLKTFETYD TFRSWTPQE MAAAGFYHTG VRLGVQCFCC SLILFGNSLR KLPIERHKKL RPECEFLQGK DVGNIGKYDI RVKRPEKMLR G GKARYHEE ...String:
MDYKDDDDKL AEHGESSEDR ISEIDYEFLP ELSALLGVDA FQVAKSQEEE EHKERMKMKK GFNSQMRSEA KRLKTFETYD TFRSWTPQE MAAAGFYHTG VRLGVQCFCC SLILFGNSLR KLPIERHKKL RPECEFLQGK DVGNIGKYDI RVKRPEKMLR G GKARYHEE EARLESFEDW PFYAHGTSPR VLSAAGFVFT GKRDTVQCFS CGGSLGNWEE GDDPWKEHAK WFPKCEFLQS KK SSEEIAQ YIQSYEGFVH VTGEHFVKSW VRRELPMVSA YCNDSVFANE ELRMDMFKDW PQESPVGVEA LVRAGFFYTG KKD IVRCFS CGGCLEKWAE GDDPMEDHIK FFPECVFLQT LKSSAEVIPT LQSQYALPEA TETTRESNHG DAAAVHSTVV DLGR SEAQW FQEARSLSEQ LRDNYTKATF RHMNLPEVCS SLGTDHLLSC DVSIISKHIS QPVQEALTIP EVFSNLNSVM CVEGE TGSG KTTFLKRIAF LWASGCCPLL YRFQLVFYLS LSSITPDQGL ANIICAQLLG AGGCISEVCL SSSIQQLQHQ VLFLLD DYS GLASLPQALH TLITKNYLSR TCLLIAVHTN RVRDIRLYLG TSLEIQEFPF YNTVSVLRKF FSHDIICVEK LIIYFID NK DLQGVYKTPL FVAAVCTDWI QNASAQDKFQ DVTLFQSYMQ YLSLKYKATA EPLQATVSSC GQLALTGLFS SCFEFNSD D LAEAGVDEDE KLTTLLMSKF TAQRLRPVYR FLGPLFQEFL AAVRLTELLS SDRQEDQDLG LYYLRQIDSP LKAINSFNI FLYYVSSHSS SKAAPTVVSH LLQLVDEKES LENMSENEDY MKLHPQTFLW FQFVRGLWLV SPESSSSFVS EHLLRLALIF AYESNTVAE CSPFILQFLR GKTLALRVLN LQYFRDHPES LLLLRSLKVS INGNKMSSYV DYSFKTYFEN LQPPAIDEEY T SAFEHISE WRRNFAQDEE IIKNYENIRP RALPDISEGY WKLSPKPCKI PKLEVQVNNT DAADQALLQV LMEVFSASQS IE FRLFNSS GFLESICPAL ELSKASVTKC SMSRLELSRA EQELLLTLPA LQSLEVSETN QLPEQLFHNL HKFLGLKELC VRL DGKPNV LSVLPREFPN LLHMEKLSIQ TSTESDLSKL VKFIQNFPNL HVFHLKCDFL SNCESLMAVL ASCKKLREIE FSGR CFEAM TFVNILPNFV SLKILNLKDQ QFPDKETSEK FAQALGSLRN LEELLVPTGD GIHQVAKLIV RQCLQLPCLR VLTFH DILD DDSVIEIARA ATSGGFQKLE NLDISMNHKI TEEGYRNFFQ ALDNLPNLQE LNICRNIPGR IQVQATTVKA LGQCVS RLP SLIRLHMLSW LLDEEDMKVI NDVKERHPQS KRLIIFWKLI VPFSPVILE

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

Concentration0.30 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5yud

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 159513
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model(PDB ID:

5yud

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6b5b

)
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-7rav:
Cryo-EM structure of the unliganded form of NLR family apoptosis inhibitory protein 5 (NAIP5)

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