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- PDB-8gtk: Crystal structure of IpaH7.8-LRR and GSDMB isoform-1 complex -

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Basic information

Entry
Database: PDB / ID: 8gtk
TitleCrystal structure of IpaH7.8-LRR and GSDMB isoform-1 complex
Components
  • GSDMB isoform-1
  • Probable E3 ubiquitin-protein ligase ipaH7.8
KeywordsIMMUNE SYSTEM / Complex / Effector / Pyroptosis
Function / homology
Function and homology information


symbiont-mediated suppression of host programmed cell death / effector-mediated activation of programmed cell death in host / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / ubiquitin-dependent protein catabolic process / host cell cytoplasm / protein ubiquitination / extracellular region
Similarity search - Function
Novel E3 ligase domain / C-terminal novel E3 ligase, LRR-interacting / LRR-containing bacterial E3 ligase, N-terminal / Type III secretion system leucine rich repeat protein / Leucine-rich repeats, bacterial type / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase ipaH7.8
Similarity search - Component
Biological speciesHomo sapiens (human)
Shigella flexneri (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsZhong, X. / Hou, Y.J. / Ding, J.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Nature / Year: 2023
Title: Structural mechanisms for regulation of GSDMB pore-forming activity.
Authors: Xiu Zhong / Huan Zeng / Zhiwei Zhou / Ya Su / Hang Cheng / Yanjie Hou / Yang She / Na Feng / Jia Wang / Feng Shao / Jingjin Ding /
Abstract: Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein, to trigger target cell pyroptosis. GSDMB and the charter gasdermin family member GSDMD have been ...Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein, to trigger target cell pyroptosis. GSDMB and the charter gasdermin family member GSDMD have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. ). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.
History
DepositionSep 8, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1May 3, 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
Revision 1.2Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GSDMB isoform-1
B: Probable E3 ubiquitin-protein ligase ipaH7.8


Theoretical massNumber of molelcules
Total (without water)72,8432
Polymers72,8432
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1750 Å2
ΔGint-0 kcal/mol
Surface area28470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)102.732, 102.732, 121.316
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

#1: Protein GSDMB isoform-1


Mass: 45002.227 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein Probable E3 ubiquitin-protein ligase ipaH7.8 / Probable RING-type E3 ubiquitin transferase ipaH7.8


Mass: 27840.521 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: ipaH7.8, CP0078, pWR501_0084, SFLP133 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P18014, RING-type E3 ubiquitin transferase

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.54 Å3/Da / Density % sol: 51.52 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 0.1M Lithium sulfate, 0.1 M Sodium Acetate pH 4.6, 0.6 M Ammonium phosphate monobasic

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL45XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Aug 1, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.1→47.3 Å / Num. obs: 13871 / % possible obs: 99.9 % / Redundancy: 14.6 % / Biso Wilson estimate: 125.32 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.063 / Rpim(I) all: 0.017 / Rrim(I) all: 0.065 / Net I/σ(I): 22.9
Reflection shellResolution: 3.1→3.31 Å / Redundancy: 15.2 % / Rmerge(I) obs: 1.046 / Mean I/σ(I) obs: 2.7 / Num. unique obs: 2483 / CC1/2: 0.934 / Rpim(I) all: 0.384 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8GTJ

8gtj


Resolution: 3.1→47.3 Å / SU ML: 0.6179 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 38.7042
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2896 1351 9.91 %
Rwork0.2657 12286 -
obs0.2681 13637 98.45 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 135.72 Å2
Refinement stepCycle: LAST / Resolution: 3.1→47.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4528 0 0 0 4528
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01024602
X-RAY DIFFRACTIONf_angle_d1.30836223
X-RAY DIFFRACTIONf_chiral_restr0.0949723
X-RAY DIFFRACTIONf_plane_restr0.0163805
X-RAY DIFFRACTIONf_dihedral_angle_d11.69561769
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.210.50131350.47451216X-RAY DIFFRACTION99.56
3.21-3.340.48921290.43181217X-RAY DIFFRACTION99.7
3.34-3.490.44971370.37721226X-RAY DIFFRACTION99.93
3.49-3.670.38631300.37131134X-RAY DIFFRACTION91.86
3.68-3.910.3861300.33731150X-RAY DIFFRACTION94.12
3.91-4.210.34671340.29171235X-RAY DIFFRACTION99.64
4.21-4.630.27341400.26541244X-RAY DIFFRACTION99.93
4.63-5.30.26961330.22911259X-RAY DIFFRACTION100
5.3-6.670.3471440.2861256X-RAY DIFFRACTION100
6.68-47.30.19251390.20821349X-RAY DIFFRACTION99.67

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