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- PDB-8aly: Cryo-EM structure of human tankyrase 2 SAM-PARP filament (G1032W ... -

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Basic information

Entry
Database: PDB / ID: 8aly
TitleCryo-EM structure of human tankyrase 2 SAM-PARP filament (G1032W mutant)
ComponentsPoly [ADP-ribose] polymerase tankyrase-2
KeywordsSIGNALING PROTEIN / Poly-ADP-ribosyltransferase / Enzyme / Polymer
Function / homology
Function and homology information


XAV939 stabilizes AXIN / NAD+ ADP-ribosyltransferase / negative regulation of telomere maintenance via telomere lengthening / protein auto-ADP-ribosylation / protein localization to chromosome, telomeric region / protein poly-ADP-ribosylation / pericentriolar material / NAD+-protein ADP-ribosyltransferase activity / positive regulation of telomere capping / Transferases; Glycosyltransferases; Pentosyltransferases ...XAV939 stabilizes AXIN / NAD+ ADP-ribosyltransferase / negative regulation of telomere maintenance via telomere lengthening / protein auto-ADP-ribosylation / protein localization to chromosome, telomeric region / protein poly-ADP-ribosylation / pericentriolar material / NAD+-protein ADP-ribosyltransferase activity / positive regulation of telomere capping / Transferases; Glycosyltransferases; Pentosyltransferases / NAD+ ADP-ribosyltransferase activity / positive regulation of telomere maintenance via telomerase / nucleotidyltransferase activity / TCF dependent signaling in response to WNT / Degradation of AXIN / Wnt signaling pathway / Regulation of PTEN stability and activity / protein polyubiquitination / positive regulation of canonical Wnt signaling pathway / nuclear envelope / chromosome, telomeric region / Ub-specific processing proteases / Golgi membrane / perinuclear region of cytoplasm / enzyme binding / nucleus / metal ion binding / cytoplasm / cytosol
Similarity search - Function
Ankyrin repeat / Ankyrin repeats (many copies) / Poly(ADP-ribose) polymerase catalytic domain / Poly(ADP-ribose) polymerase, catalytic domain / PARP catalytic domain profile. / SAM domain (Sterile alpha motif) / SAM domain profile. / Sterile alpha motif. / Sterile alpha motif domain / Sterile alpha motif/pointed domain superfamily ...Ankyrin repeat / Ankyrin repeats (many copies) / Poly(ADP-ribose) polymerase catalytic domain / Poly(ADP-ribose) polymerase, catalytic domain / PARP catalytic domain profile. / SAM domain (Sterile alpha motif) / SAM domain profile. / Sterile alpha motif. / Sterile alpha motif domain / Sterile alpha motif/pointed domain superfamily / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
Poly [ADP-ribose] polymerase tankyrase-2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsMariotti, L. / Inian, O. / Desfosses, A. / Beuron, F. / Morris, E.P. / Guettler, S.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Cancer Research UKC47521/A28286 United Kingdom
Wellcome Trust214311/Z/18/Z United Kingdom
The Institute of Cancer Research (ICR)n.a. United Kingdom
The Lister Institute of Preventive Medicinen.a. United Kingdom
CitationJournal: Nature / Year: 2022
Title: Structural basis of tankyrase activation by polymerization.
Authors: Nisha Pillay / Laura Mariotti / Mariola Zaleska / Oviya Inian / Matthew Jessop / Sam Hibbs / Ambroise Desfosses / Paul C R Hopkins / Catherine M Templeton / Fabienne Beuron / Edward P Morris ...Authors: Nisha Pillay / Laura Mariotti / Mariola Zaleska / Oviya Inian / Matthew Jessop / Sam Hibbs / Ambroise Desfosses / Paul C R Hopkins / Catherine M Templeton / Fabienne Beuron / Edward P Morris / Sebastian Guettler /
Abstract: The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT-β-catenin signalling, telomere length maintenance, Hippo ...The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT-β-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT-β-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.
History
DepositionAug 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Data collection / Database references
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 14, 2022Group: 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

Downloads & links

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Assembly

Deposited unit
E: Poly [ADP-ribose] polymerase tankyrase-2
J: Poly [ADP-ribose] polymerase tankyrase-2
I: Poly [ADP-ribose] polymerase tankyrase-2
H: Poly [ADP-ribose] polymerase tankyrase-2
G: Poly [ADP-ribose] polymerase tankyrase-2
F: Poly [ADP-ribose] polymerase tankyrase-2
D: Poly [ADP-ribose] polymerase tankyrase-2
C: Poly [ADP-ribose] polymerase tankyrase-2
B: Poly [ADP-ribose] polymerase tankyrase-2
A: Poly [ADP-ribose] polymerase tankyrase-2
O: Poly [ADP-ribose] polymerase tankyrase-2
T: Poly [ADP-ribose] polymerase tankyrase-2
S: Poly [ADP-ribose] polymerase tankyrase-2
R: Poly [ADP-ribose] polymerase tankyrase-2
Q: Poly [ADP-ribose] polymerase tankyrase-2
P: Poly [ADP-ribose] polymerase tankyrase-2
N: Poly [ADP-ribose] polymerase tankyrase-2
M: Poly [ADP-ribose] polymerase tankyrase-2
L: Poly [ADP-ribose] polymerase tankyrase-2
K: Poly [ADP-ribose] polymerase tankyrase-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)682,86240
Polymers681,55320
Non-polymers1,30820
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, also detected by mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area68570 Å2
ΔGint-879 kcal/mol
Surface area220230 Å2
MethodPISA

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Components

#1: Protein
Poly [ADP-ribose] polymerase tankyrase-2 / ADP-ribosyltransferase diphtheria toxin-like 6 / ARTD6 / Poly [ADP-ribose] polymerase 5B / Protein ...ADP-ribosyltransferase diphtheria toxin-like 6 / ARTD6 / Poly [ADP-ribose] polymerase 5B / Protein poly-ADP-ribosyltransferase tankyrase-2 / TNKS-2 / TRF1-interacting ankyrin-related ADP-ribose polymerase 2 / Tankyrase II / Tankyrase-2 / TANK2 / Tankyrase-like protein / Tankyrase-related protein


Mass: 34077.668 Da / Num. of mol.: 20 / Mutation: G1032W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TNKS2, PARP5B, TANK2, TNKL / Plasmid: pET / Details (production host): His6-MBP-Asn10-TEV tag / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21-CodonPlus (DE3)-RIL
References: UniProt: Q9H2K2, NAD+ ADP-ribosyltransferase, Transferases; Glycosyltransferases; Pentosyltransferases
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: TNKS2 SAM-PARP (867-1162) filament / Type: COMPLEX
Details: double-helical filament of human TNKS2 SAM-PARP G1032W, residues 867-1162, N-terminal vector-derived SNA tripeptide, 20 protomers in refined structure
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 34 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21-CodonPlus (DE3)-RIL / Plasmid: pET
Buffer solutionpH: 7.5
Details: After brief incubation on the grid, the sample was washed 10 times with water to gradually lower the salt concentration and improve sample contrast.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris bufferTris-HCl1
2500 mMsodium chlorideNaCl1
310 mMbeta-mercaptoethanolbeta-ME1
SpecimenConc.: 0.86 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The PARP domain is inactivated by a G1032W mutation.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1140INTEGRATINGGATAN K2 SUMMIT (4k x 4k)
2140GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3.08CTF correction
7UCSF Chimera1.14model fitting
8Coot0.9model fitting
12RELION3.08classification
13RELION3.083D reconstruction
14PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -52.3 ° / Axial rise/subunit: 13.6 Å / Axial symmetry: D1
Particle selectionNum. of particles selected: 188925
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139880 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingB value: 7.41 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: FSC
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
15NWG

5nwg

IB1
25JRT

5jrt

A1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00542280
ELECTRON MICROSCOPYf_angle_d0.54756940
ELECTRON MICROSCOPYf_dihedral_angle_d19.0415680
ELECTRON MICROSCOPYf_chiral_restr0.0446040
ELECTRON MICROSCOPYf_plane_restr0.0047260

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