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- PDB-7z6o: X-Ray studies of Ku70/80 reveal the binding site for IP6 -

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Basic information

Entry
Database: PDB / ID: 7z6o
TitleX-Ray studies of Ku70/80 reveal the binding site for IP6
Components
  • DNA (5'-D(*GP*TP*TP*TP*TP*TP*AP*GP*TP*TP*TP*AP*T)-3')
  • DNA (5'-D(P*AP*AP*AP*TP*AP*AP*AP*CP*TP*AP*AP*AP*AP*AP*C)-3')
  • X-ray repair cross-complementing protein 5
  • X-ray repair cross-complementing protein 6
KeywordsDNA BINDING PROTEIN / DNA Repair / Double-Strand Break / NHEJ
Function / homology
Function and homology information


Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / cellular response to X-ray ...Ku70:Ku80 complex / negative regulation of t-circle formation / DNA end binding / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / cellular response to X-ray / nuclear telomere cap complex / double-strand break repair via classical nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / positive regulation of neurogenesis / recombinational repair / regulation of telomere maintenance / protein localization to chromosome, telomeric region / U3 snoRNA binding / cellular hyperosmotic salinity response / 2-LTR circle formation / telomeric DNA binding / hematopoietic stem cell proliferation / positive regulation of protein kinase activity / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / hematopoietic stem cell differentiation / ATP-dependent activity, acting on DNA / telomere maintenance via telomerase / neurogenesis / telomere maintenance / activation of innate immune response / DNA helicase activity / cyclin binding / cellular response to leukemia inhibitory factor / small-subunit processome / Nonhomologous End-Joining (NHEJ) / enzyme activator activity / cellular response to gamma radiation / protein-DNA complex / double-strand break repair via nonhomologous end joining / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / double-strand break repair / double-stranded DNA binding / scaffold protein binding / secretory granule lumen / DNA recombination / transcription regulator complex / ficolin-1-rich granule lumen / damaged DNA binding / chromosome, telomeric region / transcription cis-regulatory region binding / ribonucleoprotein complex / innate immune response / negative regulation of DNA-templated transcription / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / positive regulation of DNA-templated transcription / protein-containing complex binding / nucleolus / positive regulation of transcription by RNA polymerase II / protein-containing complex / ATP hydrolysis activity / DNA binding / RNA binding / extracellular region / nucleoplasm / ATP binding / nucleus / membrane / plasma membrane / cytosol
Similarity search - Function
Ku70; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm ...Ku70; Chain: A; domain 4 / Ku70; Chain: A; domain 4 - #10 / Ku70, bridge and pillars domain superfamily / : / Ku70 / Ku, C-terminal / Ku, C-terminal domain superfamily / Ku C terminal domain like / Ku80 / Ku70/Ku80 C-terminal arm / Ku70/Ku80 C-terminal arm / Ku70/Ku80, N-terminal alpha/beta / Ku70/Ku80 N-terminal alpha/beta domain / Ku70/Ku80 beta-barrel domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily / SAP domain superfamily / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / von Willebrand factor, type A domain / von Willebrand factor (vWF) type A domain / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / DNA / DNA (> 10) / X-ray repair cross-complementing protein 6 / X-ray repair cross-complementing protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.7 Å
AuthorsVarela, P.F. / Charbonnier, J.B.
Funding support France, 4items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0026 France
Agence Nationale de la Recherche (ANR)ANR-17-CE12-0020 France
Agence Nationale de la Recherche (ANR)ANR-21-CE12-0019-01 France
Agence Nationale de la Recherche (ANR)ANR-10-INBS-0005 France
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.
Authors: Antonia Kefala Stavridi / Amandine Gontier / Vincent Morin / Philippe Frit / Virginie Ropars / Nadia Barboule / Carine Racca / Sagun Jonchhe / Michael J Morten / Jessica Andreani / Alexey ...Authors: Antonia Kefala Stavridi / Amandine Gontier / Vincent Morin / Philippe Frit / Virginie Ropars / Nadia Barboule / Carine Racca / Sagun Jonchhe / Michael J Morten / Jessica Andreani / Alexey Rak / Pierre Legrand / Alexa Bourand-Plantefol / Steven W Hardwick / Dimitri Y Chirgadze / Paul Davey / Taiana Maia De Oliveira / Eli Rothenberg / Sebastien Britton / Patrick Calsou / Tom L Blundell / Paloma F Varela / Amanda K Chaplin / Jean-Baptiste Charbonnier /
Abstract: The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by ...The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.
History
DepositionMar 14, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Database references / Category: citation / citation_author
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
Revision 1.2Dec 6, 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

Downloads & links

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Assembly

Deposited unit
A: X-ray repair cross-complementing protein 6
B: X-ray repair cross-complementing protein 5
C: DNA (5'-D(*GP*TP*TP*TP*TP*TP*AP*GP*TP*TP*TP*AP*T)-3')
D: DNA (5'-D(P*AP*AP*AP*TP*AP*AP*AP*CP*TP*AP*AP*AP*AP*AP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,2495
Polymers169,5894
Non-polymers6601
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23580 Å2
ΔGint-142 kcal/mol
Surface area49910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.146, 125.146, 319.700
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"

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Components

#1: Protein X-ray repair cross-complementing protein 6 / 5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP- ...5'-deoxyribose-5-phosphate lyase Ku70 / 5'-dRP lyase Ku70 / 70 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 1 / ATP-dependent DNA helicase II 70 kDa subunit / CTC box-binding factor 75 kDa subunit / CTCBF / DNA repair protein XRCC6 / Lupus Ku autoantigen protein p70 / Ku70 / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 6


Mass: 69945.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC6, G22P1 / Production host: unidentified baculovirus
References: UniProt: P12956, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases
#2: Protein X-ray repair cross-complementing protein 5 / 86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase ...86 kDa subunit of Ku antigen / ATP-dependent DNA helicase 2 subunit 2 / ATP-dependent DNA helicase II 80 kDa subunit / CTC box-binding factor 85 kDa subunit / CTCBF / DNA repair protein XRCC5 / Ku80 / Ku86 / Lupus Ku autoantigen protein p86 / Nuclear factor IV / Thyroid-lupus autoantigen / TLAA / X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining)


Mass: 82812.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues from loop 173-176 are modeled as alanines because of their side chains missing density
Source: (gene. exp.) Homo sapiens (human) / Gene: XRCC5, G22P2 / Production host: unidentified baculovirus
References: UniProt: P13010, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: DNA chain DNA (5'-D(*GP*TP*TP*TP*TP*TP*AP*GP*TP*TP*TP*AP*T)-3')


Mass: 6506.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*AP*AP*AP*TP*AP*AP*AP*CP*TP*AP*AP*AP*AP*AP*C)-3')


Mass: 10325.685 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: 4.26 Å3/Da / Density % sol: 71.14 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 24% PEG3350, 200mM Na2SO4, 100mM BTP, 5 mM b-mercaptoethanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.980101 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Mar 6, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.980101 Å / Relative weight: 1
ReflectionResolution: 3.7→48.3 Å / Num. obs: 31855 / % possible obs: 99.7 % / Redundancy: 20.1 % / Biso Wilson estimate: 115.41 Å2 / CC1/2: 0.996 / Net I/σ(I): 4.2
Reflection shellResolution: 3.7→3.79 Å / Redundancy: 18.1 % / Num. unique obs: 2327 / CC1/2: 0.047 / % possible all: 98.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
BUSTERrefinement
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
STARANISOdata scaling
PHASERphasing
PDB_EXTRACT3.27data extraction
MxCuBEdata collection
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1jey

1jey


Resolution: 3.7→48.3 Å / SU ML: 0.4565 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 33.2182
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2713 655 5.13 %RANDOM
Rwork0.2179 12123 --
obs0.2205 12778 40.07 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 209.23 Å2
Refinement stepCycle: LAST / Resolution: 3.7→48.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8326 571 36 0 8933
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00169167
X-RAY DIFFRACTIONf_angle_d0.422412487
X-RAY DIFFRACTIONf_chiral_restr0.03751388
X-RAY DIFFRACTIONf_plane_restr0.0031503
X-RAY DIFFRACTIONf_dihedral_angle_d15.52483565
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.7-3.980.3495350.2512659X-RAY DIFFRACTION11.07
3.98-4.380.3244530.21251006X-RAY DIFFRACTION16.98
4.38-5.020.26181010.21541608X-RAY DIFFRACTION26.97
5.02-6.320.3171290.24662648X-RAY DIFFRACTION43.65
6.32-48.30.2583370.21166202X-RAY DIFFRACTION98.42
Refinement TLS params.Method: refined / Origin x: -63.975453812 Å / Origin y: 35.5152228262 Å / Origin z: -22.2608290425 Å
111213212223313233
T0.856468692823 Å20.169510545482 Å2-0.0435792236997 Å2-1.03490201675 Å20.129541976192 Å2--0.925183122047 Å2
L1.15673668071 °20.229107169244 °2-0.844962461391 °2-0.766785546849 °2-0.345059236178 °2--3.24078379337 °2
S0.0504988700613 Å °0.0337951317413 Å °-0.0880806209914 Å °0.153987020555 Å °-0.0810636953763 Å °0.204647421444 Å °0.203066365165 Å °-0.635322084481 Å °0.0148302495437 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsLabel asym-IDLabel seq-ID
1X-RAY DIFFRACTION1{ A|1-34 }A - E1
2X-RAY DIFFRACTION1{ E|1-34 }

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