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- PDB-8sl0: Structure of a bacterial gasdermin slinky-like oligomer -

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Basic information

Entry
Database: PDB / ID: 8sl0
TitleStructure of a bacterial gasdermin slinky-like oligomer
ComponentsGasdermin bGSDM
KeywordsIMMUNE SYSTEM / viral mimicry / gasdermin / caspase / autoinhibition / pyroptosis / bats / immunity / cell death
Function / homologydefense response to virus / plasma membrane / cytoplasm / Gasdermin bGSDM
Function and homology information
Biological speciesVitiosangium sp. GDMCC 1.1324 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsJohnson, A.G. / Mayer, M.L. / Kranzusch, P.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM146250-01 United States
Citation
Journal: Nature / Year: 2024
Title: Structure and assembly of a bacterial gasdermin pore.
Authors: Alex G Johnson / Megan L Mayer / Stefan L Schaefer / Nora K McNamara-Bordewick / Gerhard Hummer / Philip J Kranzusch /
Abstract: In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores have revealed the ...In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
#1: Journal: bioRxiv / Year: 2023
Title: Structure and assembly of a bacterial gasdermin pore.
Authors: Alex G Johnson / Megan L Mayer / Stefan L Schaefer / Nora K McNamara-Bordewick / Gerhard Hummer / Philip J Kranzusch /
Abstract: In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores reveal the functions ...In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores reveal the functions and architectures of 24-33 protomers assemblies, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of a bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
History
DepositionApr 20, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_DOI ..._citation.journal_abbrev / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 3, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Revision 1.3May 1, 2024Group: 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: Gasdermin bGSDM


Theoretical massNumber of molelcules
Total (without water)25,5661
Polymers25,5661
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, assembly is derived from a membrane pore sample which was visualized by EM on liposomes and was biochemically demonstrated to perforate membranes
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Gasdermin bGSDM / bGSDM / Bacterial gasdermin


Mass: 25566.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vitiosangium sp. GDMCC 1.1324 (bacteria)
Gene: DAT35_31115, Ga0334635_1658 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A2T4VDM4

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Vitiosangium bGSDM in an active slinky-like oligomeric conformation
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vitiosangium sp. GDMCC 1.1324 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET
Buffer solutionpH: 7.5
Details: 150 mM NaCl, 20 mM HEPES-HOH (pH 7.5), 5.15 mM DDMAB
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMHEPES-KOH buffer (pH 7.5)1
35.16 mMN-Dodecyl-N,N-(dimethylammonio)butyrate1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 51.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8156

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: SerialEM / Version: 3.8.6 / Category: image acquisition
CTF correctionType: NONE
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132403 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0031764
ELECTRON MICROSCOPYf_angle_d0.5682384
ELECTRON MICROSCOPYf_dihedral_angle_d4.214242
ELECTRON MICROSCOPYf_chiral_restr0.046274
ELECTRON MICROSCOPYf_plane_restr0.004312

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