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- PDB-9ebs: Cryo-EM structure of USP1-UAF1-Ubiquitin in complex with TNG348 -

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Basic information

Entry
Database: PDB / ID: 9ebs
TitleCryo-EM structure of USP1-UAF1-Ubiquitin in complex with TNG348
Components
  • Ubiquitin
  • Ubiquitin carboxyl-terminal hydrolase 1
  • WD repeat-containing protein 48
KeywordsHYDROLASE / deubiquitination / PCNA / allosteric inhibitor
Function / homology
Function and homology information


regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / skin development / symbiont entry into host cell via disruption of host cell envelope ...regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / skin development / symbiont entry into host cell via disruption of host cell envelope / virus tail / homeostasis of number of cells / protein deubiquitination / embryonic organ development / single fertilization / positive regulation of double-strand break repair via homologous recombination / regulation of DNA repair / response to UV / Fanconi Anemia Pathway / positive regulation of epithelial cell proliferation / ubiquitin binding / Recognition of DNA damage by PCNA-containing replication complex / skeletal system development / double-strand break repair via homologous recombination / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein stability / multicellular organism growth / late endosome / peptidase activity / single-stranded DNA binding / double-stranded DNA binding / spermatogenesis / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / lysosome / Ub-specific processing proteases / cysteine-type endopeptidase activity / DNA repair / intracellular membrane-bounded organelle / DNA damage response / proteolysis / DNA binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / 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 ...WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / 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. / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Pectin lyase fold / Pectin lyase fold/virulence factor / Papain-like cysteine peptidase superfamily / WD domain, G-beta repeat / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
: / : / Ubiquitin carboxyl-terminal hydrolase 1 / Tail fiber / WD repeat-containing protein 48
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWhittington, D.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Mol Cancer Ther / Year: 2025
Title: Characterization of TNG348: A Selective, Allosteric USP1 Inhibitor That Synergizes with PARP Inhibitors in Tumors with Homologous Recombination Deficiency.
Authors: Antoine Simoneau / Charlotte B Pratt / Hsin-Jung Wu / Shreya S Rajeswaran / Charlotte Grace Comer / Sirimas Sudsakorn / Wenhai Zhang / Shangtao Liu / Samuel R Meier / Ashley H Choi / Tenzing ...Authors: Antoine Simoneau / Charlotte B Pratt / Hsin-Jung Wu / Shreya S Rajeswaran / Charlotte Grace Comer / Sirimas Sudsakorn / Wenhai Zhang / Shangtao Liu / Samuel R Meier / Ashley H Choi / Tenzing Khendu / Hannah Stowe / Binzhang Shen / Douglas A Whittington / Yingnan Chen / Yi Yu / William D Mallender / Tianshu Feng / Jannik N Andersen / John P Maxwell / Scott Throner /
Abstract: Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the ...Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the treatment of BRCA1/2-mutant cancers, potentially including patients whose tumors have primary or acquired resistance to PARP inhibitors (PARPi). In this study, we present a comprehensive characterization of TNG348, an allosteric, selective, and reversible inhibitor of USP1. TNG348 induces dose-dependent accumulation of ubiquitinated protein substrates both in vitro and in vivo. CRISPR screens show that TNG348 exerts its antitumor effect by disrupting the translesion synthesis pathway of DNA damage tolerance through RAD18-dependent ubiquitinated PCNA. Although TNG348 and PARPi share the ability to selectively kill HRD tumor cells, CRISPR screens reveal that TNG348 and PARPi do so through discrete mechanisms. Particularly, knocking out PARP1 causes resistance to PARPi but sensitizes cells to TNG348 treatment. Consistent with these findings, combination of TNG348 with PARPi leads to synergistic antitumor effects in HRD tumors, resulting in tumor growth inhibition and regression in multiple mouse xenograft tumor models. Importantly, our data on human cancer models further show that the addition of TNG348 to PARPi treatment can overcome acquired PARPi resistance in vivo. Although the clinical development of TNG348 has been discontinued because of unexpected liver toxicity in patients (NCT06065059), the present data provide preclinical and mechanistic support for the continued exploration of USP1 as a drug target for the treatment of patients with BRCA1/2-mutant or HRD cancers.
History
DepositionNov 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation_author.name / _em_admin.last_update
Revision 1.2May 14, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Ubiquitin
D: WD repeat-containing protein 48
E: Ubiquitin carboxyl-terminal hydrolase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)175,0426
Polymers174,2353
Non-polymers8063
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules CDE

#1: Protein Ubiquitin


Mass: 8519.778 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: vinyl sulfone modification at C-terminus / Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG47
#2: Protein WD repeat-containing protein 48 / USP1-associated factor 1 / WD repeat endosomal protein / p80


Mass: 77309.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal Flag tag / Source: (gene. exp.) Homo sapiens (human) / Gene: WDR48, KIAA1449, UAF1 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TAF3
#3: Protein Ubiquitin carboxyl-terminal hydrolase 1 / Deubiquitinating enzyme 1 / hUBP / Ubiquitin thioesterase 1 / Ubiquitin-specific-processing protease 1


Mass: 88406.344 Da / Num. of mol.: 1 / Mutation: G670A,G671A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: USP1 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O94782, ubiquitinyl hydrolase 1

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Non-polymers , 3 types, 3 molecules

#4: Chemical ChemComp-A1A4Y / 3-(methanesulfonyl)propan-1-amine


Mass: 137.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H11NO2S
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-A1BHF / 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-({4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenyl}methyl)-7-(2,2,2-trifluoroethyl)-7,9-dihydro-8H-purin-8-imine


Mass: 603.522 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H23F6N9O / Feature type: SUBJECT OF INVESTIGATION

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Details

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: USP1-UAF1-Ub complex with TNG348 / Type: COMPLEX
Details: USP1-UAF1 co-expressed and purified; Ub-VS then used to covalently modify USP1
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.174 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMsodium chloride1
35 %glycerol1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 51.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6175
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv3.3particle selection
4cryoSPARCv3.3CTF correction
9cryoSPARCv3.3initial Euler assignment
10cryoSPARCv3.3final Euler assignment
11cryoSPARCv3.3classification
12cryoSPARCv3.33D reconstruction
13PHENIX1.18_3845model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3700674
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151407 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
15CVO

5cvo

A5CVOA1
27AY2

7ay2

B7AY2B2
37AY2

7ay2

C7AY2C2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 73.21 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00257782
ELECTRON MICROSCOPYf_angle_d0.464710528
ELECTRON MICROSCOPYf_chiral_restr0.04171208
ELECTRON MICROSCOPYf_plane_restr0.00361313
ELECTRON MICROSCOPYf_dihedral_angle_d7.82481015

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