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- PDB-7pag: The pore conformation of lymphocyte perforin -

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Basic information

Entry
Database: PDB / ID: 7pag
TitleThe pore conformation of lymphocyte perforin
ComponentsPerforin-1
KeywordsIMMUNE SYSTEM / Pore forming protein perforin
Function / homology
Function and homology information


immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / protein transmembrane transport / immunological synapse formation / wide pore channel activity / protein import / positive regulation of killing of cells of another organism / defense response to tumor cell ...immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / protein transmembrane transport / immunological synapse formation / wide pore channel activity / protein import / positive regulation of killing of cells of another organism / defense response to tumor cell / immunological synapse / protein secretion / endosome lumen / protein homooligomerization / T cell mediated cytotoxicity / circadian rhythm / cytoplasmic vesicle / defense response to virus / killing of cells of another organism / calcium ion binding / extracellular space / membrane / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Perforin-1, C2 domain / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain ...Perforin-1, C2 domain / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / C2 domain superfamily
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsIvanova, M.E. / Lukoyanova, N. / Malhotra, S. / Topf, M. / Trapani, J.A. / Voskoboinik, I. / Saibil, H.R.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust209250/Z/17/Z United Kingdom
Wellcome Trust208398/Z/17/Z United Kingdom
European Research Council (ERC)294408 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: The pore conformation of lymphocyte perforin.
Authors: Marina E Ivanova / Natalya Lukoyanova / Sony Malhotra / Maya Topf / Joseph A Trapani / Ilia Voskoboinik / Helen R Saibil /
Abstract: Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with ...Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo-electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central β sheet to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.
History
DepositionJul 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: Perforin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,1685
Polymers60,8271
Non-polymers3414
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Monomeric perforin oligomerises into arcs and rings on the membrane in the presence of Ca2+ at 37 degrees, electron microscopy, Negative stain EM was performed to ...Evidence: assay for oligomerization, Monomeric perforin oligomerises into arcs and rings on the membrane in the presence of Ca2+ at 37 degrees, electron microscopy, Negative stain EM was performed to confirm the presence of perforin oligomers
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area410 Å2
ΔGint-22 kcal/mol
Surface area31220 Å2

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Components

#1: Protein Perforin-1 / / P1 / Cytolysin / Lymphocyte pore-forming protein


Mass: 60827.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Prf1, Pfp / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P10820
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Oligomerised mouse Perforin in its inserted state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R2/2
VitrificationCryogen 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 FIELDBright-field microscopy
Image recordingElectron dose: 49.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229789
Details: 229,789 particles were symmetry expanded with C22 symmetry giving the final number of particles 5,055,358
Symmetry type: POINT

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