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- EMDB-20554: Cryo-EM structure of DDB1-DCAF15 bound to E7820 and the second RR... -

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Basic information

Entry
Database: EMDB / ID: EMD-20554
TitleCryo-EM structure of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39
Map data
Sample
  • Complex: Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39
    • Protein or peptide: DNA Damage-Binding Protein 1
    • Protein or peptide: DDB1 And CUL4 Associated Factor 15
    • Protein or peptide: RNA Binding Motif Protein 39
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsFaust T / Yoon H / Nowak RP / Donovan KA / Li Z / Cai Q / Eleuteri NA / Zhang T / Gray NS / Fischer ES
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteR01CA214608 United States
CitationJournal: Nat Chem Biol / Year: 2020
Title: Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15.
Authors: Tyler B Faust / Hojong Yoon / Radosław P Nowak / Katherine A Donovan / Zhengnian Li / Quan Cai / Nicholas A Eleuteri / Tinghu Zhang / Nathanael S Gray / Eric S Fischer /
Abstract: The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically ...The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 Å, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.
History
DepositionAug 2, 2019-
Header (metadata) releaseAug 14, 2019-
Map releaseNov 20, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0073
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0073
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20554.png

Downloads & links

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Map

FileDownload / File: emd_20554.map.gz / Format: CCP4 / Size: 85.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.0073 / Movie #1: 0.0073
Minimum - Maximum-0.007559877 - 0.02655806
Average (Standard dev.)0.0000064177 (±0.000996567)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions282282282
Spacing282282282
CellA=B=C: 300.048 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z282282282
origin x/y/z0.0000.0000.000
length x/y/z300.048300.048300.048
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ172172172
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS282282282
D min/max/mean-0.0080.0270.000

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Supplemental data

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Mask #1

Fileemd_20554_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Histogram (log scale)

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Additional map: #1

Fileemd_20554_additional.map
Projections & Slices
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Additional map: #1

Fileemd_20554_additional_1.map
Projections & Slices
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Half map: #1

Fileemd_20554_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_20554_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39

EntireName: Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39
Components
  • Complex: Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39
    • Protein or peptide: DNA Damage-Binding Protein 1
    • Protein or peptide: DDB1 And CUL4 Associated Factor 15
    • Protein or peptide: RNA Binding Motif Protein 39

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Supramolecule #1: Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39

SupramoleculeName: Complex of DDB1-DCAF15 bound to E7820 and the second RRM of RBM39
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Compound: E7820; Chemical Name: 3-Cyano-N-(3-cyano-4-methyl-1H-indol-7-yl)benzenesulfonamide
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 211 KDa

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Macromolecule #1: DNA Damage-Binding Protein 1

MacromoleculeName: DNA Damage-Binding Protein 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSSHHHHHH SAVDENLYFQ GGGRMSYNYV VTAQKPTAVN GCVTGHFTSA EDLNLLIAKN TRLEIYVVTA EGLRPVKEVG MYGKIAVMEL FRPKGESKDL LFILTAKYNA CILEYKQSGE SIDIITRAHG NVQDRIGRPS ETGIIGIIDP ECRMIGLRLY DGLFKVIPLD ...String:
MGSSHHHHHH SAVDENLYFQ GGGRMSYNYV VTAQKPTAVN GCVTGHFTSA EDLNLLIAKN TRLEIYVVTA EGLRPVKEVG MYGKIAVMEL FRPKGESKDL LFILTAKYNA CILEYKQSGE SIDIITRAHG NVQDRIGRPS ETGIIGIIDP ECRMIGLRLY DGLFKVIPLD RDNKELKAFN IRLEELHVID VKFLYGCQAP TICFVYQDPQ GRHVKTYEVS LREKEFNKGP WKQENVEAEA SMVIAVPEPF GGAIIIGQES ITYHNGDKYL AIAPPIIKQS TIVCHNRVDP NGSRYLLGDM EGRLFMLLLE KEEQMDGTVT LKDLRVELLG ETSIAECLTY LDNGVVFVGS RLGDSQLVKL NVDSNEQGSY VVAMETFTNL GPIVDMCVVD LERQGQGQLV TCSGAFKEGS LRIIRNGIGI HEHASIDLPG IKGLWPLRSD PNRETDDTLV LSFVGQTRVL MLNGEEVEET ELMGFVDDQQ TFFCGNVAHQ QLIQITSASV RLVSQEPKAL VSEWKEPQAK NISVASCNSS QVVVAVGRAL YYLQIHPQEL RQISHTEMEH EVACLDITPL GDSNGLSPLC AIGLWTDISA RILKLPSFEL LHKEMLGGEI IPRSILMTTF ESSHYLLCAL GDGALFYFGL NIETGLLSDR KKVTLGTQPT VLRTFRSLST TNVFACSDRP TVIYSSNHKL VFSNVNLKEV NYMCPLNSDG YPDSLALANN STLTIGTIDE IQKLHIRTVP LYESPRKICY QEVSQCFGVL SSRIEVQDTS GGTTALRPSA STQALSSSVS SSKLFSSSTA PHETSFGEEV EVHNLLIIDQ HTFEVLHAHQ FLQNEYALSL VSCKLGKDPN TYFIVGTAMV YPEEAEPKQG RIVVFQYSDG KLQTVAEKEV KGAVYSMVEF NGKLLASINS TVRLYEWTTE KELRTECNHY NNIMALYLKT KGDFILVGDL MRSVLLLAYK PMEGNFEEIA RDFNPNWMSA VEILDDDNFL GAENAFNLFV CQKDSAATTD EERQHLQEVG LFHLGEFVNV FCHGSLVMQN LGETSTPTQG SVLFGTVNGM IGLVTSLSES WYNLLLDMQN RLNKVIKSVG KIEHSFWRSF HTERKTEPAT GFIDGDLIES FLDISRPKMQ EVVANLQYDD GSGMKREATA DDLIKVVEEL TRIH

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Macromolecule #2: DDB1 And CUL4 Associated Factor 15

MacromoleculeName: DDB1 And CUL4 Associated Factor 15 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDWSHPQFEK SAVDENLYFQ GGCGRMAPSS KSERNSGAGS GGGGPGGAGG KRAAGRRREH VLKQLERVKI SGQLSPRLFR KLPPRVCVSL KNIVDEDFLY AGHIFLGFSK CGRYVLSYTS SSGDDDFSFY IYHLYWWEFN VHSKLKLVRQ VRLFQDEEIY SDLYLTVCEW ...String:
MDWSHPQFEK SAVDENLYFQ GGCGRMAPSS KSERNSGAGS GGGGPGGAGG KRAAGRRREH VLKQLERVKI SGQLSPRLFR KLPPRVCVSL KNIVDEDFLY AGHIFLGFSK CGRYVLSYTS SSGDDDFSFY IYHLYWWEFN VHSKLKLVRQ VRLFQDEEIY SDLYLTVCEW PSDASKVIVF GFNTRSANGM LMNMMMMSDE NHRDIYVSTV AVPPPGRCAA CQDASRAHPG DPNAQCLRHG FMLHTKYQVV YPFPTFQPAF QLKKDQVVLL NTSYSLVACA VSVHSAGDRS FCQILYDHST CPLAPASPPE PQSPELPPAL PSFCPEAAPA RSSGSPEPSP AIAKAKEFVA DIFRRAKEAK GGVPEEARPA LCPGPSGSRC RAHSEPLALC GETAPRDSPP ASEAPASEPG YVNYTKLYYV LESGEGTEPE DELEDDKISL PFVVTDLRGR NLRPMRERTA VQGQYLTVEQ LTLDFEYVIN EVIRHDATWG HQFCSFSDYD IVILEVCPET NQVLINIGLL LLAFPSPTEE GQLRPKTYHT SLKVAWDLNT GIFETVSVGD LTEVKGQTSG SVWSSYRKSC VDMVMKWLVP ESSGRYVNRM TNEALHKGCS LKVLADSERY TWIVL

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Macromolecule #3: RNA Binding Motif Protein 39

MacromoleculeName: RNA Binding Motif Protein 39 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MGSSHHHHHH SAVDENLYFQ GGCGRGSAGP MRLYVGSLHF NITEDMLRGI FEPFGRIESI QLMMDSETGR SKGYGFITFS DSECAKKALE QLNGFELAGR PMKVGHVTER TDA

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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

Concentration0.075 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClSodium chloridesodium chloride
2.0 mMTCEPtris(2-carboxyethyl)phosphine
20.0 uME7820N-(3-cyano-4-methyl-1H-indol-7-yl)-3-cyanobenzene-sulfonamide

Details: Gel filtration buffer was made fresh the day of the purification
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 286 K / Instrument: LEICA EM GP / Details: Blotted for 3 seconds before plunging..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 1457 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 318187
Details: Initial 2D classification resulted in a set of 318,187 particles
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1), RELION (ver. 3.0.4)) / Details: Standard CTF correction inside Relion
Startup modelType of model: OTHER
Details: Relion 3.0.4 was used to generate an initial model from the data.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.0.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.4) / Number images used: 68324
FSC plot (resolution estimation)

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