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- PDB-9yxy: Crystal structure of EEPD1 EEP domain dimer at pH 5.5 -

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Basic information

Entry
Database: PDB / ID: 9yxy
TitleCrystal structure of EEPD1 EEP domain dimer at pH 5.5
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
IMIDAZOLE / Endonuclease/exonuclease/phosphatase family domain-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsShen, R. / Arvai, A.S. / Brosey, C.A. / Tsai, C. / 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 27, 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)66,6505
Polymers66,4272
Non-polymers2233
Water3,387188
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SAXS, cross-linking, assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1670 Å2
ΔGint1 kcal/mol
Surface area22790 Å2
Unit cell
Length a, b, c (Å)38.943, 116.320, 121.483
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails (eV)
d_1ens_1(chain "A" and (resid 254 through 308 or resid 310...
d_2ens_1(chain "B" and (resid 254 through 308 or resid 310...

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11THRTHRLEULEUAA254 - 30813 - 67
d_12LYSLYSLEULEUAA310 - 40869 - 167
d_13SERSERLYSLYSAA428 - 481187 - 240
d_14PROPROPROPROAA483 - 516242 - 275
d_15ILEILEGLUGLUAA518 - 541277 - 300
d_21THRTHRLEULEUBB254 - 30813 - 67
d_22LYSLYSLYSLYSBB310 - 48169 - 240
d_23PROPROPROPROBB483 - 516242 - 275
d_24ILEILEGLUGLUBB518 - 541277 - 300

NCS oper: (Code: givenMatrix: (-0.999942148393, 0.00834066360127, -0.00679214229115), (0.00175876532386, -0.496173900072, -0.868221381695), (-0.0106116262066, -0.868183099477, 0.496130526345)Vector: ...NCS oper: (Code: given
Matrix: (-0.999942148393, 0.00834066360127, -0.00679214229115), (0.00175876532386, -0.496173900072, -0.868221381695), (-0.0106116262066, -0.868183099477, 0.496130526345)
Vector: 31.8132630469, 21.1083841807, 11.5357631775)

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Components

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


Mass: 33213.562 Da / Num. of mol.: 2
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
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 188 / Source method: isolated from a natural source / Formula: H2O
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: 2.07 Å3/Da / Density % sol: 40.61 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 0.1 M Bis-Tris, pH 5.5, 25% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 17, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2→38.24 Å / Num. obs: 37041 / % possible obs: 96.68 % / Redundancy: 13.6 % / Biso Wilson estimate: 38.55 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.1613 / Rpim(I) all: 0.04504 / Rrim(I) all: 0.1677 / Net I/σ(I): 11.85
Reflection shellResolution: 2→2.05 Å / Redundancy: 14.2 % / Rmerge(I) obs: 3.158 / Mean I/σ(I) obs: 0.86 / Num. unique obs: 2799 / CC1/2: 0.399 / CC star: 0.755 / Rpim(I) all: 0.8626 / Rrim(I) all: 3.276 / % possible all: 94.9

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419refinement
XDS20210323data reduction
Aimless0.8.2data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→38.24 Å / SU ML: 0.2593 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.2251
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2323 1805 4.87 %
Rwork0.1944 35225 -
obs0.1963 37030 96.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 55.64 Å2
Refinement stepCycle: LAST / Resolution: 2→38.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4098 0 15 188 4301
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00364253
X-RAY DIFFRACTIONf_angle_d0.70125775
X-RAY DIFFRACTIONf_chiral_restr0.0473626
X-RAY DIFFRACTIONf_plane_restr0.0067744
X-RAY DIFFRACTIONf_dihedral_angle_d13.62971541
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 1.22103582154 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.050.35051530.31092641X-RAY DIFFRACTION94.9
2.05-2.110.32121420.28592704X-RAY DIFFRACTION99.86
2.11-2.180.32531450.28372646X-RAY DIFFRACTION95.78
2.18-2.260.34031340.25862710X-RAY DIFFRACTION98.51
2.26-2.350.29271180.23582572X-RAY DIFFRACTION92.92
2.35-2.460.27421360.21912730X-RAY DIFFRACTION97.45
2.46-2.590.26881230.21372744X-RAY DIFFRACTION98.39
2.59-2.750.23651580.21112704X-RAY DIFFRACTION98.45
2.75-2.960.30521200.2012654X-RAY DIFFRACTION94.1
2.96-3.260.20271410.17632752X-RAY DIFFRACTION97.7
3.26-3.730.18781130.17112701X-RAY DIFFRACTION95.23
3.73-4.70.1731560.14822827X-RAY DIFFRACTION99.17
4.7-38.240.23891660.19742840X-RAY DIFFRACTION94.71

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