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- PDB-9k2x: Cryo-EM structure of USP7:DNMT1 complex; open conformation -

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Basic information

Entry
Database: PDB / ID: 9k2x
TitleCryo-EM structure of USP7:DNMT1 complex; open conformation
Components
  • DNA (cytosine-5)-methyltransferase 1
  • Ubiquitin carboxyl-terminal hydrolase 7
KeywordsSTRUCTURAL PROTEIN / DNA methylation / deubiquitination
Function / homology
Function and homology information


chromosomal DNA methylation maintenance following DNA replication / : / epigenetic programming of gene expression / regulation of telomere capping / regulation of establishment of protein localization to telomere / DNA-methyltransferase activity / negative regulation of vascular associated smooth muscle cell apoptotic process / monoubiquitinated protein deubiquitination / cellular response to bisphenol A / regulation of retrograde transport, endosome to Golgi ...chromosomal DNA methylation maintenance following DNA replication / : / epigenetic programming of gene expression / regulation of telomere capping / regulation of establishment of protein localization to telomere / DNA-methyltransferase activity / negative regulation of vascular associated smooth muscle cell apoptotic process / monoubiquitinated protein deubiquitination / cellular response to bisphenol A / regulation of retrograde transport, endosome to Golgi / DNA (cytosine-5-)-methyltransferase / deubiquitinase activity / DNA (cytosine-5-)-methyltransferase activity / SUMOylation of DNA methylation proteins / DNA alkylation repair / : / female germ cell nucleus / STAT3 nuclear events downstream of ALK signaling / methyl-CpG binding / K48-linked deubiquitinase activity / symbiont-mediated disruption of host cell PML body / DNA methylation-dependent constitutive heterochromatin formation / negative regulation of gene expression via chromosomal CpG island methylation / : / lncRNA binding / protein deubiquitination / negative regulation of gluconeogenesis / pericentric heterochromatin / positive regulation of vascular associated smooth muscle cell proliferation / Nuclear events stimulated by ALK signaling in cancer / negative regulation of TORC1 signaling / transcription-coupled nucleotide-excision repair / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / Regulation of PTEN localization / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / DNA methylation / regulation of signal transduction by p53 class mediator / replication fork / PRC2 methylates histones and DNA / Defective pyroptosis / cellular response to amino acid stimulus / promoter-specific chromatin binding / regulation of circadian rhythm / NoRC negatively regulates rRNA expression / PML body / regulation of protein stability / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / p53 binding / Regulation of TP53 Degradation / rhythmic process / chromosome / methylation / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Ub-specific processing proteases / protein stabilization / nuclear body / protein ubiquitination / negative regulation of gene expression / cysteine-type endopeptidase activity / DNA-templated transcription / positive regulation of gene expression / negative regulation of transcription by RNA polymerase II / protein-containing complex / mitochondrion / proteolysis / DNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
DMAP1-binding Domain / DMAP1-binding Domain / DMAP1-binding domain / DMAP1-binding domain profile. / DNA (cytosine-5)-methyltransferase 1-like / DNA (cytosine-5)-methyltransferase 1, replication foci domain / Cytosine specific DNA methyltransferase replication foci domain / : / Ubiquitin carboxyl-terminal hydrolase 7, ICP0-binding domain / ICP0-binding domain of Ubiquitin-specific protease 7 ...DMAP1-binding Domain / DMAP1-binding Domain / DMAP1-binding domain / DMAP1-binding domain profile. / DNA (cytosine-5)-methyltransferase 1-like / DNA (cytosine-5)-methyltransferase 1, replication foci domain / Cytosine specific DNA methyltransferase replication foci domain / : / Ubiquitin carboxyl-terminal hydrolase 7, ICP0-binding domain / ICP0-binding domain of Ubiquitin-specific protease 7 / DNA methylase, C-5 cytosine-specific, conserved site / C-5 cytosine-specific DNA methylases C-terminal signature. / Ubiquitin carboxyl-terminal hydrolase, C-terminal / Ubiquitin-specific protease C-terminal / MATH domain / CXXC zinc finger domain / Zinc finger, CXXC-type / Zinc finger CXXC-type profile. / : / DNA methylase, C-5 cytosine-specific, active site / C-5 cytosine-specific DNA methylases active site. / C-5 cytosine-specific DNA methylase (Dnmt) domain profile. / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / C-5 cytosine methyltransferase / C-5 cytosine-specific DNA methylase / TRAF-like / Bromo adjacent homology domain / BAH domain / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Papain-like cysteine peptidase superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
DNA (cytosine-5)-methyltransferase 1 / Ubiquitin carboxyl-terminal hydrolase 7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsNakamura, N. / Arita, K.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP18H02392 Japan
Japan Society for the Promotion of Science (JSPS)JP19H05294 Japan
Japan Society for the Promotion of Science (JSPS)JP19H05741 Japan
Japan Society for the Promotion of Science (JSPS)19H05285 Japan
Japan Society for the Promotion of Science (JSPS)21H00272 Japan
Japan Society for the Promotion of Science (JSPS)24K02001 Japan
CitationJournal: Structure / Year: 2025
Title: Structures of USP7 in active and inactive states bound to DNMT1 revealed by cryo-EM.
Authors: Nao Nakamura / Sae Yoshimi / Amika Kikuchi / Hiroki Onoda / Satomi Kori / Makoto Nakanishi / Atsuya Nishiyama / Kyohei Arita /
Abstract: The ubiquitin signal generated by UHRF1 is essential for DNA methylation maintenance by recruiting DNA methyltransferase 1 (DNMT1) to hemimethylated DNA through strong binding of its replication foci ...The ubiquitin signal generated by UHRF1 is essential for DNA methylation maintenance by recruiting DNA methyltransferase 1 (DNMT1) to hemimethylated DNA through strong binding of its replication foci targeting sequence (RFTS) domain to ubiquitinated histone H3. The ubiquitin-specific protease 7 (USP7) forms a complex with DNMT1 and removes ubiquitin from H3. However, it remains unknown how USP7 deubiquitinates ubiquitinated H3 upon strong binding of the DNMT1 RFTS domain. Here, we show the activation mechanism of USP7 by combining biochemical and structural studies. USP7 is inactive toward ubiquitinated H3 in complex with the RFTS domain. However, when complexed with DNMT1, USP7 efficiently deubiquitinates ubiquitinated H3. Cryogenic electron microscopy (cryo-EM) single particle analysis revealed that USP7 bound to DNMT1 undergoes an open (inactive) and closed (active) conformational transition. Our findings provide mechanistic insights into the activation of USP7 upon binding to DNMT1 and contribute to a better understanding of the deubiquitination process in DNA methylation maintenance.
History
DepositionOct 18, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 25, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquitin carboxyl-terminal hydrolase 7
B: DNA (cytosine-5)-methyltransferase 1


Theoretical massNumber of molelcules
Total (without water)272,4442
Polymers272,4442
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ubiquitin carboxyl-terminal hydrolase 7 / Deubiquitinating enzyme 7 / Herpesvirus-associated ubiquitin-specific protease / Ubiquitin ...Deubiquitinating enzyme 7 / Herpesvirus-associated ubiquitin-specific protease / Ubiquitin thioesterase 7 / Ubiquitin-specific-processing protease 7


Mass: 128884.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: USP7, HAUSP / Production host: Escherichia coli (E. coli) / References: UniProt: Q93009, ubiquitinyl hydrolase 1
#2: Protein DNA (cytosine-5)-methyltransferase 1 / Dnmt1 / CXXC-type zinc finger protein 9 / DNA methyltransferase HsaI / DNA MTase HsaI / M.HsaI / MCMT


Mass: 143559.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DNMT1, AIM, CXXC9, DNMT / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P26358, DNA (cytosine-5-)-methyltransferase
Has ligand of interestY
Has protein modificationN

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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: DNMT1:USP7 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 62.918 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73679 / Symmetry type: POINT
RefinementHighest resolution: 3.75 Å / Cross valid method: NONE
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412666
ELECTRON MICROSCOPYf_angle_d0.85317131
ELECTRON MICROSCOPYf_dihedral_angle_d4.7881694
ELECTRON MICROSCOPYf_chiral_restr0.0491832
ELECTRON MICROSCOPYf_plane_restr0.0062250

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