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- EMDB-24389: Cryo-EM structure of the unliganded form of NLR family apoptosis ... -

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Basic information

Entry
Database: EMDB / ID: EMD-24389
TitleCryo-EM structure of the unliganded form of NLR family apoptosis inhibitory protein 5 (NAIP5) with partial LRR domain
Map data
Sample
  • Cell: Pre-liganded form of NAIP5
    • Protein or peptide: NLR family) apoptosis inhibitory protein 5 (NAIP5)
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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 3, 2021-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateMar 8, 2023-
Current statusMar 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24389.png

Downloads & links

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Map

FileDownload / File: emd_24389.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0694 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.086
Minimum - Maximum-0.0017185382 - 1.9882016
Average (Standard dev.)0.0011015633 (±0.025026996)
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_24389_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_24389_half_map_2.map
Projections & Slices
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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: NLR family) apoptosis inhibitory protein 5 (NAIP5)

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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: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: NLR family) apoptosis inhibitory protein 5 (NAIP5)

MacromoleculeName: NLR family) apoptosis inhibitory protein 5 (NAIP5) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKL AEHGESSEDR ISEIDYEFLP ELSALLGVDA FQVAKSQEEE EHKERMKMKK GFNSQMRSEA KRLKTFETYD TFRSWTPQEM AAAGFYHTGV RLGVQCFCCS LILFGNSLRK LPIERHKKLR PECEFLQGKD VGNIGKYDIR VKRPEKMLRG GKARYHEEEA ...String:
MDYKDDDDKL AEHGESSEDR ISEIDYEFLP ELSALLGVDA FQVAKSQEEE EHKERMKMKK GFNSQMRSEA KRLKTFETYD TFRSWTPQEM AAAGFYHTGV RLGVQCFCCS LILFGNSLRK LPIERHKKLR PECEFLQGKD VGNIGKYDIR VKRPEKMLRG GKARYHEEEA RLESFEDWPF YAHGTSPRV LSAAGFVFTG KRDTVQCFSC GGSLGNWEEG DDPWKEHAKW FPKCEFLQSK KSSEEIAQYI QSYEGFVHV TGEHFVKSWV RRELPMVSAY CNDSVFANEE LRMDMFKDWP QESPVGVEAL V RAGFFYTG KKDIVRCFSC GGCLEKWAEG DDPMEDHIKF FPECVFLQTL KSSAEVIPTL QS QYALPEA TETTRESNHG DAAAVHSTVV DLGRSEAQWF QEARSLSEQL RDNYTKATFR HMN LPEVCS SLGTDHLLSC DVSIISKHIS QPVQEALTIP EVFSNLNSVM CVEGETGSGK TTFL KRIAF LWASGCCPLL YRFQLVFYLS LSSITPDQGL ANIICAQLLG AGGCISEVCL SSSIQ QLQH QVLFLLDDYS GLASLPQALH TLITKNYLSR TCLLIAVHTN RVRDIRLYLG TSLEIQ EFP FYNTVSVLRK FFSHDIICVE KLIIYFIDNK DLQGVYKTPL FVAAVCTDWI QNASAQD KF QDVTLFQSYM QYLSLKYKAT AEPLQATVSS CGQLALTGLF SSCFEFNSDD LAEAGVDE D EKLTTLLMSK FTAQRLRPVY RFLGPLFQEF LAAVRLTELL SSDRQEDQDL GLYYLRQID SPLKAINSFN IFLYYVSSHS SSKAAPTVVS HLLQLVDEKE SLENMSENED YMKLHPQTFL WFQFVRGLW LVSPESSSSF VSEHLLRLAL IFAYESNTVA ECSPFILQFL RGKTLALRVL N LQYFRDHP ESLLLLRSLK VSINGNKMSS YVDYSFKTYF ENLQPPAIDE EYTSAFEHIS EW RRNFAQD EEIIKNYENI RPRALPDISE GYWKLSPKPC KIPKLEVQVN NTDAADQALL QVL MEVFSA SQSIEFRLFN SSGFLESICP ALELSKASVT KCSMSRLELS RAEQELLLTL PALQ SLEVS ETNQLPEQLF HNLHKFLGLK ELCVRLDGKP NVLSVLPREF PNLLHMEKLS IQTST ESDL SKLVKFIQNF PNLHVFHLKC DFLSNCESLM AVLASCKKLR EIEFSGRCFE AMTFVN ILP NFVSLKILNL KDQQFPDKET SEKFAQALGS LRNLEELLVP TGDGIHQVAK LIVRQCL QL PCLRVLTFHD ILDDDSVIEI ARAATSGGFQ KLENLDISMN HKITEEGYRN FFQALDNL P NLQELNICRN IPGRIQVQAT TVKALGQCVS RLPSLIRLHM LSWLLDEEDM KVINDVKER HPQSKRLIIF WKLIVPFSPV ILE

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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
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN 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: 2.5 µm / Nominal defocus min: 1.0 µm
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: 3.6 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

RefinementProtocol: OTHER

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