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- EMDB-50317: USP1 bound to ML323 and ubiquitin conjugated to FANCD2 (ordered s... -

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Basic information

Entry
Database: EMDB / ID: EMD-50317
TitleUSP1 bound to ML323 and ubiquitin conjugated to FANCD2 (ordered subset, focused refinement)
Map dataGlobally sharpened map (B-factor 81.5)
Sample
  • Complex: USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2
    • Protein or peptide: Polyubiquitin-C
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
  • Ligand: ZINC ION
  • Ligand: 5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phenyl]methyl]pyrimidin-4-amine
  • Ligand: water
KeywordsINHIBITOR / DEUBIQUITINASE / COMPLEX / ENZYME-SUBSTRATE / HYDROLASE
Function / homology
Function and homology information


positive regulation of error-prone translesion synthesis / monoubiquitinated protein deubiquitination / protein deubiquitination / regulation of DNA repair / response to UV / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / IRAK2 mediated activation of TAK1 complex ...positive regulation of error-prone translesion synthesis / monoubiquitinated protein deubiquitination / protein deubiquitination / regulation of DNA repair / response to UV / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / FLT3 signaling by CBL mutants / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Prevention of phagosomal-lysosomal fusion / Glycogen synthesis / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Endosomal Sorting Complex Required For Transport (ESCRT) / TICAM1,TRAF6-dependent induction of TAK1 complex / Membrane binding and targetting of GAG proteins / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Negative regulation of FLT3 / Constitutive Signaling by NOTCH1 HD Domain Mutants / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of FZD by ubiquitination / TICAM1-dependent activation of IRF3/IRF7 / NOTCH2 Activation and Transmission of Signal to the Nucleus / p75NTR recruits signalling complexes / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / TRAF6-mediated induction of TAK1 complex within TLR4 complex / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / Regulation of innate immune responses to cytosolic DNA / NF-kB is activated and signals survival / InlA-mediated entry of Listeria monocytogenes into host cells / Regulation of pyruvate metabolism / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Pexophagy / Activated NOTCH1 Transmits Signal to the Nucleus / Regulation of PTEN localization / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Translesion synthesis by POLK / Downregulation of TGF-beta receptor signaling / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Translesion synthesis by POLI / PINK1-PRKN Mediated Mitophagy / IKK complex recruitment mediated by RIP1 / Regulation of activated PAK-2p34 by proteasome mediated degradation / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Gap-filling DNA repair synthesis and ligation in GG-NER / Josephin domain DUBs / InlB-mediated entry of Listeria monocytogenes into host cell / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / skeletal system development / APC/C:Cdc20 mediated degradation of Securin / Regulation of NF-kappa B signaling / SCF-beta-TrCP mediated degradation of Emi1 / Asymmetric localization of PCP proteins / TCF dependent signaling in response to WNT / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / activated TAK1 mediates p38 MAPK activation / TNFR2 non-canonical NF-kB pathway / Vpu mediated degradation of CD4 / Negative regulators of DDX58/IFIH1 signaling / NOTCH3 Activation and Transmission of Signal to the Nucleus / Assembly of the pre-replicative complex / Degradation of DVL / Deactivation of the beta-catenin transactivating complex / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Regulation of signaling by CBL / Dectin-1 mediated noncanonical NF-kB signaling / positive regulation of receptor signaling pathway via JAK-STAT / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Fanconi Anemia Pathway / Negative regulation of FGFR3 signaling / Hh mutants are degraded by ERAD / Degradation of AXIN / Peroxisomal protein import / Activation of NF-kappaB in B cells / Regulation of TNFR1 signaling / Degradation of GLI1 by the proteasome / Recognition of DNA damage by PCNA-containing replication complex / Negative regulation of FGFR2 signaling / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Stabilization of p53 / EGFR downregulation / Hedgehog ligand biogenesis
Similarity search - Function
Ubiquitin specific peptidase 1 / : / 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 ...Ubiquitin specific peptidase 1 / : / 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 / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin carboxyl-terminal hydrolase 1 / Polyubiquitin-C
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsRennie ML / Gundogdu M / Walden H
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/W025256/1 United Kingdom
CitationJournal: J Med Chem / Year: 2024
Title: Structural and Biochemical Insights into the Mechanism of Action of the Clinical USP1 Inhibitor, KSQ-4279.
Authors: Martin Luke Rennie / Mehmet Gundogdu / Connor Arkinson / Steven Liness / Sheelagh Frame / Helen Walden /
Abstract: DNA damage triggers cell signaling cascades that mediate repair. This signaling is frequently dysregulated in cancers. The proteins that mediate this signaling are potential targets for therapeutic ...DNA damage triggers cell signaling cascades that mediate repair. This signaling is frequently dysregulated in cancers. The proteins that mediate this signaling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small-molecule inhibitors already in clinical trials. Here, we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel-like pocket in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.
History
DepositionMay 15, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50317.png

Downloads & links

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Map

FileDownload / File: emd_50317.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally sharpened map (B-factor 81.5)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.72064763 - 1.2695192
Average (Standard dev.)0.000030750994 (±0.025799224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 339.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50317_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_50317_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #3

Fileemd_50317_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2

EntireName: USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2
Components
  • Complex: USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2
    • Protein or peptide: Polyubiquitin-C
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
  • Ligand: ZINC ION
  • Ligand: 5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phenyl]methyl]pyrimidin-4-amine
  • Ligand: water

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Supramolecule #1: USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2

SupramoleculeName: USP1 BOUND TO ML323 AND UBIQUITIN CONJUGATED TO FANCD2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Polyubiquitin-C

MacromoleculeName: Polyubiquitin-C / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.875125 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GPGSMQIFVK TLTGKTITLE VEPSDTIENV KAKIQDKEGI PPDQQRLIFA GKQLEDGRTL SDYNIQKEST LHLVLRLRGG

UniProtKB: Polyubiquitin-C

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Macromolecule #2: Ubiquitin carboxyl-terminal hydrolase 1

MacromoleculeName: Ubiquitin carboxyl-terminal hydrolase 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: ubiquitinyl hydrolase 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 88.390273 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMPGVIPSES NGLSRGSPSK KNRLSLKFFQ KKETKRALDF TDSQENEEKA SEYRASEIDQ VVPAAQSSPI NCEKRENLLP FVGLNNLGN TSYLNSILQV LYFCPGFKSG VKHLFNIISR KKEALKDEAN QKDKGNCKED SLASYELICS LQSLIISVEQ L QASFLLNP ...String:
GMPGVIPSES NGLSRGSPSK KNRLSLKFFQ KKETKRALDF TDSQENEEKA SEYRASEIDQ VVPAAQSSPI NCEKRENLLP FVGLNNLGN TSYLNSILQV LYFCPGFKSG VKHLFNIISR KKEALKDEAN QKDKGNCKED SLASYELICS LQSLIISVEQ L QASFLLNP EKYTDELATQ PRRLLNTLRE LNPMYEGYLQ HDAQEVLQCI LGNIQETCQL LKKEEVKNVA ELPTKVEEIP HP KEEMNGI NSIEMDSMRH SEDFKEKLPK GNGKRKSDTE FGNMKKKVKL SKEHQSLEEN QRQTRSKRKA TSDTLESPPK IIP KYISEN ESPRPSQKKS RVKINWLKSA TKQPSILSKF CSLGKITTNQ GVKGQSKENE CDPEEDLGKC ESDNTTNGCG LESP GNTVT PVNVNEVKPI NKGEEQIGFE LVEKLFQGQL VLRTRCLECE SLTERREDFQ DISVPVQEDE LSKVEESSEI SPEPK TEMK TLRWAISQFA SVERIVGEDK YFCENCHHYT EAERSLLFDK MPEVITIHLK CFAASGLEFD CYGGGLSKIN TPLLTP LKL SLEEWSTKPT NDSYGLFAVV MHSGITISSG HYTASVKVTD LNSLELDKGN FVVDQMCEIG KPEPLNEEEA RGVVENY ND EEVSIRVGGN TQPSKVLNKK NVEAIGLLAA QKSKADYELY NKASNPDKVA STAFAENRNS ETSDTTGTHE SDRNKESS D QTGINISGFE NKISYVVQSL KEYEGKWLLF DDSEVKVTEE KDFLNSLSPS TSPTSTPYLL FYKKL

UniProtKB: Ubiquitin carboxyl-terminal hydrolase 1

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: 5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phe...

MacromoleculeName: 5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phenyl]methyl]pyrimidin-4-amine
type: ligand / ID: 4 / Number of copies: 1 / Formula: JDA
Molecular weightTheoretical: 384.477 Da
Chemical component information

ChemComp-JDA:
5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phenyl]methyl]pyrimidin-4-amine

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 29 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV / Details: blotting for 3.0 seconds.
Details9.3 uM USP1-UAF1, 1.8 uM FANCI-FANCD2Ub, 2.2 uM dsDNA (61 base-pairs), 18 uM ML323

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Details2x binning
Particle selectionNumber selected: 6716868
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 124414
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7zh4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Overall B value: 81.5
Output model

PDB-9fcj:
USP1 bound to ML323 and ubiquitin conjugated to FANCD2 (ordered subset, focused refinement)

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