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- PDB-9ysf: Crystal structure of EEPD1 EEP domain dimer with Mg(II) -

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Basic information

Entry
Database: PDB / ID: 9ysf
TitleCrystal structure of EEPD1 EEP domain dimer with Mg(II)
ComponentsEndonuclease/exonuclease/phosphatase family domain-containing protein 1
KeywordsDNA BINDING PROTEIN / Endonuclease/Exonuclease/Phosphatase Fold / Dimerization / Replication stress response
Function / homology
Function and homology information


catalytic activity / positive regulation of cholesterol efflux / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / DNA repair / DNA binding / plasma membrane
Similarity search - Function
Competence protein ComEA, helix-hairpin-helix domain / : / Helix-hairpin-helix motif / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / RuvA domain 2-like / Endonuclease/exonuclease/phosphatase superfamily / Helix-hairpin-helix DNA-binding motif, class 1 / Helix-hairpin-helix DNA-binding motif class 1
Similarity search - Domain/homology
Endonuclease/exonuclease/phosphatase family domain-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.59 Å
AuthorsShen, R. / Arvai, A.S. / Brosey, C.A. / Tsutakawa, S.E. / Tainer, J.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P01 CA092584 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 CA220430 United States
CitationJournal: Nucleic Acids Res / Year: 2026
Title: EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks.
Authors: Runze Shen / Altaf H Sarker / Yue Chen / Min Liu / Sunetra Roy / Andrew S Arvai / Albino Bacolla / Zamal Ahmed / Panagiotis Katsonis / Michal Hammel / Isao Kuraoka / Miaw-Sheue Tsai / ...Authors: Runze Shen / Altaf H Sarker / Yue Chen / Min Liu / Sunetra Roy / Andrew S Arvai / Albino Bacolla / Zamal Ahmed / Panagiotis Katsonis / Michal Hammel / Isao Kuraoka / Miaw-Sheue Tsai / Corydon Irie / Lukas Webb / Olivier Lichtarge / Chi-Lin Tsai / Susan E Tsutakawa / Katharina Schlacher / John A Tainer /
Abstract: Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 ...Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH)2] domains to clamp double-stranded (ds) DNA at reversed DNA replication forks for fork protection. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable "wrist" enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication fork intermediates but alone lacks nuclease activity due to loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Furthermore, cancer bioinformatics support oxidative damage-dependent EEPD1 association as a significant modulator of overall patient survival. Collective findings uncover unexpected EEP dimer and fork protection function in clamping, not cleaving, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.
History
DepositionOct 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2026Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2026Group: Database references
Category: citation / citation_author / pdbx_database_related
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endonuclease/exonuclease/phosphatase family domain-containing protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,80210
Polymers32,3121
Non-polymers4909
Water2,972165
1
A: Endonuclease/exonuclease/phosphatase family domain-containing protein 1
hetero molecules

A: Endonuclease/exonuclease/phosphatase family domain-containing protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,60320
Polymers64,6232
Non-polymers98018
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,y,-z1
Unit cell
Length a, b, c (Å)87.318, 48.210, 73.695
Angle α, β, γ (deg.)90.000, 110.018, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11A-823-

HOH

21A-845-

HOH

31A-857-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Endonuclease/exonuclease/phosphatase family domain-containing protein 1


Mass: 32311.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EEPD1, KIAA1706 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta2 / References: UniProt: Q7L9B9

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Non-polymers , 5 types, 174 molecules

#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 165 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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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: 2.26 Å3/Da / Density % sol: 45.46 %
Crystal growTemperature: 288 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 0.1 M Tris, pH 8.5, 0.3 M magnesium chloride, 25% w/v PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-1 / Wavelength: 0.97946 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 28, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 1.59→38.72 Å / Num. obs: 37207 / % possible obs: 95.35 % / Redundancy: 6.7 % / Biso Wilson estimate: 26.21 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.04806 / Rpim(I) all: 0.01976 / Rrim(I) all: 0.05206 / Net I/σ(I): 14.21
Reflection shellResolution: 1.59→1.63 Å / Redundancy: 4.9 % / Rmerge(I) obs: 1.274 / Num. unique obs: 2119 / CC1/2: 0.68 / CC star: 0.9 / Rpim(I) all: 0.6345 / Rrim(I) all: 1.429 / % possible all: 70.58

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419refinement
XDS20210323data reduction
Aimless0.7.7data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.59→38.72 Å / SU ML: 0.1987 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.3769
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2043 1796 4.84 %
Rwork0.166 35327 -
obs0.1677 37123 95.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 50.23 Å2
Refinement stepCycle: LAST / Resolution: 1.59→38.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2052 0 28 165 2245
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00972138
X-RAY DIFFRACTIONf_angle_d1.00062893
X-RAY DIFFRACTIONf_chiral_restr0.0661311
X-RAY DIFFRACTIONf_plane_restr0.012369
X-RAY DIFFRACTIONf_dihedral_angle_d17.9593776
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.59-1.630.40621030.38772006X-RAY DIFFRACTION70.58
1.63-1.680.34651070.33422316X-RAY DIFFRACTION81.75
1.68-1.740.34651530.27352728X-RAY DIFFRACTION96.55
1.74-1.80.2581490.24592837X-RAY DIFFRACTION99.43
1.8-1.870.27671390.21222769X-RAY DIFFRACTION99.08
1.87-1.950.22211510.17292795X-RAY DIFFRACTION99.03
1.95-2.060.20091470.14882806X-RAY DIFFRACTION98.66
2.06-2.190.2171460.14882787X-RAY DIFFRACTION98.13
2.19-2.360.19731720.14492809X-RAY DIFFRACTION99.83
2.36-2.590.1951470.14792831X-RAY DIFFRACTION99.63
2.59-2.970.1841280.14612839X-RAY DIFFRACTION98.67
2.97-3.740.17531050.14812890X-RAY DIFFRACTION99.04
3.74-38.720.19311490.16732914X-RAY DIFFRACTION98.9
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.39986491051-0.341282163599-1.586138226982.601312576730.4279188362444.25146047477-0.0347681686056-0.177248808597-0.2474345475690.191019408192-0.004933606229240.1080189331720.414603595120.03358636527190.08648655746550.416091082888-0.0192627648346-0.04598370842870.268103434060.02254753539940.19479658921427.650730532136.040565898814.7774425982
22.42020620843-0.370548805789-0.3224701801893.509435483150.9174173437534.347993084990.0114758074792-0.05957329408710.05070118738590.0702782488732-0.08039397833630.143969332166-0.0391349753657-0.3593184360980.08799041950360.325066898623-0.00590584511265-0.05766455937230.2601666149410.01182556453290.16061674435220.725092764842.296662001811.543703624
38.414044363485.59531730366-2.675988572468.55949893661-0.285027166873.60548275936-0.609845690660.04688331662871.16035265766-0.5310880391410.5993307722671.87111201264-1.44563070132-0.1861019736020.0578277589371.709190245520.4717829105380.09148996515020.9913589568640.2036768548291.1966718859816.747895001261.985109412422.057056459
42.07742287842-1.03701897084-1.537597560091.912365613931.538512031394.66901136696-0.148961057909-0.3437945717190.1301561627270.2343601493140.196374965523-0.0811470272085-0.009301650593420.121894451818-0.05613029243530.4166123369830.0248934415291-0.05821765450930.2871829968260.001245160957540.17633823338930.337586780445.374609740422.8188452914
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 253 through 291 )253 - 2911 - 39
22chain 'A' and (resid 292 through 424 )292 - 42440 - 144
33chain 'A' and (resid 425 through 439 )425 - 439145 - 159
44chain 'A' and (resid 440 through 542 )440 - 542160 - 262

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