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- PDB-7rav: Cryo-EM structure of the unliganded form of NLR family apoptosis ... -

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Basic information

Entry
Database: PDB / ID: 7rav
TitleCryo-EM structure of the unliganded form of NLR family apoptosis inhibitory protein 5 (NAIP5)
ComponentsBaculoviral IAP repeat-containing protein 1e
KeywordsIMMUNE SYSTEM / pre-liganded NAIP5 / inflammasome / innate immunity / host-pathogen interaction
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 / apoptotic process / negative regulation of apoptotic process / ATP binding / metal ion binding
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / NLRC4, helical domain / NLRC4 helical domain / NOD2, winged helix domain / NOD2 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 / NOD2, winged helix domain / NOD2 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
ADENOSINE-5'-TRIPHOSPHATE / Baculoviral IAP repeat-containing protein 1e
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsPaidimuddala, B. / Cao, J. / Xie, Q. / Wu, H. / Zhang, L.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Category: citation / citation_author
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.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Baculoviral IAP repeat-containing protein 1e
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,6352
Polymers161,1281
Non-polymers5071
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 1e / Neuronal apoptosis inhibitory protein 5


Mass: 161128.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Naip5, Birc1e, Naip-rs3 / Plasmid: pCMV-flag-NAIP5 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9R016
#2: 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
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: pre-liganded form of NAIP5 / Type: CELL / Entity ID: #1 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: pCMV-Flag-NAIP5
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Coot0.9.4.1 ELmodel fitting
12cryoSPARC3D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159513 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
15YUD

5yud

1
26B5B

6b5b

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 91.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00496353
ELECTRON MICROSCOPYf_angle_d0.81488586
ELECTRON MICROSCOPYf_chiral_restr0.0502943
ELECTRON MICROSCOPYf_plane_restr0.00531090
ELECTRON MICROSCOPYf_dihedral_angle_d10.6404849

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