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- EMDB-19567: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G7... -

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Entry
Database: EMDB / ID: EMD-19567
TitleClosed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
Map dataCryo-EM structure of the (NEDD8)-CRL2VHL-MZ1-Brd4BD2 ternary complex primed for catalysis with UBE2R1-Ub.
Sample
  • Complex: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
KeywordsE3 ligase / Cullin / PROTAC / BET bromodomain / LIGASE
Function / homology
Function and homology information


regulation of cellular response to hypoxia / cellular response to camptothecin / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / negative regulation of beige fat cell differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / negative regulation of mitophagy / (E3-independent) E2 ubiquitin-conjugating enzyme / cullin-RING ubiquitin ligase complex ...regulation of cellular response to hypoxia / cellular response to camptothecin / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / negative regulation of beige fat cell differentiation / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / negative regulation of mitophagy / (E3-independent) E2 ubiquitin-conjugating enzyme / cullin-RING ubiquitin ligase complex / regulation of xenophagy / target-directed miRNA degradation / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / elongin complex / regulation of cell cycle process / neural crest cell differentiation / Replication of the SARS-CoV-1 genome / RNA polymerase II transcription initiation surveillance / positive regulation of protein autoubiquitination / protein neddylation / transcription elongation factor activity / regulation of BMP signaling pathway / mitochondrion transport along microtubule / NEDD8 ligase activity / positive regulation of protein monoubiquitination / regulation of mitophagy / negative regulation of response to oxidative stress / regulation of centrosome duplication / protein K27-linked ubiquitination / VCB complex / Cul5-RING ubiquitin ligase complex / regulation of TOR signaling / E2 ubiquitin-conjugating enzyme / ubiquitin-ubiquitin ligase activity / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / SUMOylation of ubiquitinylation proteins / Cul2-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / negative regulation of DNA-templated DNA replication / regulation of mitotic cytokinesis / Cul3-RING ubiquitin ligase complex / regulation of DNA damage checkpoint / negative regulation of transcription elongation by RNA polymerase II / negative regulation of type I interferon production / regulation of miRNA-mediated gene silencing / regulation of natural killer cell activation / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / histone H4K8ac reader activity / Prolactin receptor signaling / regulation of cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / regulation of stem cell population maintenance / Cul4B-RING E3 ubiquitin ligase complex / RNA polymerase II C-terminal domain binding / histone H3K27ac reader activity / negative regulation of DNA damage checkpoint / TGF-beta receptor signaling activates SMADs / P-TEFb complex binding / histone H3K9ac reader activity / ubiquitin conjugating enzyme activity / ubiquitin ligase complex scaffold activity / negative regulation of adipose tissue development / histone H4 reader activity / regulation of proteolysis / regulation of cellular response to stress / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / histone H4K5ac reader activity / histone H4K12ac reader activity / limb development / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / host-mediated suppression of viral transcription / histone H4K16ac reader activity / regulation of postsynapse assembly / DNA replication initiation / protein monoubiquitination / negative regulation of signal transduction / anatomical structure morphogenesis / cullin family protein binding / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / positive regulation of T-helper 17 cell lineage commitment / Tat-mediated elongation of the HIV-1 transcript / cellular response to interferon-beta / Formation of HIV-1 elongation complex containing HIV-1 Tat / regulation of DNA-templated DNA replication initiation / centrosome duplication / Formation of HIV elongation complex in the absence of HIV Tat / cell morphogenesis / positive regulation of G2/M transition of mitotic cell cycle / cilium assembly / ubiquitin-like ligase-substrate adaptor activity / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / intrinsic apoptotic signaling pathway / ribosome-associated ubiquitin-dependent protein catabolic process / signal transduction in response to DNA damage
Similarity search - Function
Nedd8-like ubiquitin / von Hippel-Lindau disease tumour suppressor, beta/alpha domain / von Hippel-Lindau disease tumour suppressor, alpha domain / von Hippel-Lindau disease tumour suppressor, beta domain / VHL superfamily / von Hippel-Lindau disease tumour suppressor, alpha domain superfamily / von Hippel-Lindau disease tumour suppressor, beta domain superfamily / VHL beta domain / VHL box domain / : ...Nedd8-like ubiquitin / von Hippel-Lindau disease tumour suppressor, beta/alpha domain / von Hippel-Lindau disease tumour suppressor, alpha domain / von Hippel-Lindau disease tumour suppressor, beta domain / VHL superfamily / von Hippel-Lindau disease tumour suppressor, alpha domain superfamily / von Hippel-Lindau disease tumour suppressor, beta domain superfamily / VHL beta domain / VHL box domain / : / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Elongin-C / Elongin B / : / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin, N-terminal / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin alpha solenoid domain / Cullin / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Brdt, bromodomain, repeat I / Brdt, bromodomain, repeat II / NET domain superfamily / NET domain profile. / : / NET domain / Bromodomain extra-terminal - transcription regulation / SKP1/BTB/POZ domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Bromodomain, conserved site / Bromodomain signature. / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Bromodomain-containing protein 4 / Polyubiquitin-B / von Hippel-Lindau disease tumor suppressor / Ubiquitin-conjugating enzyme E2 R1 / E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-C / Elongin-B / Ubiquitin-like protein NEDD8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsCiulli A / Crowe C / Nakasone MA
Funding supportEuropean Union, United Kingdom, Switzerland, 5 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
Medical Research Council (MRC, United Kingdom) United Kingdom
Innovative Medicines Initiative Switzerland
Wellcome Trust United Kingdom
Cancer Research UK United Kingdom
Citation
Journal: Sci Adv / Year: 2024
Title: Mechanism of degrader-targeted protein ubiquitinability.
Authors: Charlotte Crowe / Mark A Nakasone / Sarah Chandler / Conner Craigon / Gajanan Sathe / Michael H Tatham / Nikolai Makukhin / Ronald T Hay / Alessio Ciulli /
Abstract: Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived ...Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4 toward UBE2R1-ubiquitin, and Lys at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4, with cellular degradation and ubiquitinomics confirming the importance of Lys and nearby Lys/Lys, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.
#1: Journal: Biorxiv / Year: 2024
Title: Mechanism of degrader-targeted protein ubiquitinability
Authors: Crowe C / Nakasone MA / Chandler S / Tatham MH / Makukhin N / Hay RT / Ciulli A
History
DepositionFeb 5, 2024-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19567.png

Downloads & links

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Map

FileDownload / File: emd_19567.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the (NEDD8)-CRL2VHL-MZ1-Brd4BD2 ternary complex primed for catalysis with UBE2R1-Ub.
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 396. Å		0.83 Å/pix.
x 480 pix.
= 396. Å		0.83 Å/pix.
x 480 pix.
= 396. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.8583815 - 1.2370622
Average (Standard dev.)-0.00006806529 (±0.011533733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19567_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_19567_half_map_2.map
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Sample components

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Entire : Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G7...

EntireName: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
Components
  • Complex: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub

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Supramolecule #1: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G7...

SupramoleculeName: Closed crosslinked structure of (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 190 kDa/nm

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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: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 149823
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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