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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 / negative regulation of beige fat cell differentiation / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / cullin-RING-type E3 NEDD8 transferase / (E3-independent) E2 ubiquitin-conjugating enzyme / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / Cul7-RING ubiquitin ligase complex / cellular response to chemical stress ...regulation of cellular response to hypoxia / negative regulation of beige fat cell differentiation / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / cullin-RING-type E3 NEDD8 transferase / (E3-independent) E2 ubiquitin-conjugating enzyme / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / Cul7-RING ubiquitin ligase complex / cellular response to chemical stress / transcription elongation factor activity / target-directed miRNA degradation / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / elongin complex / protein K27-linked ubiquitination / positive regulation of protein autoubiquitination / RNA polymerase II transcription initiation surveillance / protein neddylation / hypothalamus gonadotrophin-releasing hormone neuron development / Replication of the SARS-CoV-1 genome / female meiosis I / NEDD8 ligase activity / positive regulation of protein monoubiquitination / VCB complex / fat pad development / negative regulation of response to oxidative stress / mitochondrion transport along microtubule / Cul5-RING ubiquitin ligase complex / intracellular membraneless organelle / ubiquitin-ubiquitin ligase activity / SCF ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / E2 ubiquitin-conjugating enzyme / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / female gonad development / SUMOylation of ubiquitinylation proteins / seminiferous tubule development / negative regulation of mitophagy / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Prolactin receptor signaling / histone H4K8ac reader activity / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / histone H3K9ac reader activity / RNA polymerase II C-terminal domain binding / histone H3K27ac reader activity / ubiquitin conjugating enzyme activity / TGF-beta receptor signaling activates SMADs / P-TEFb complex binding / male meiosis I / negative regulation of DNA damage checkpoint / histone H4 reader activity / negative regulation of transcription elongation by RNA polymerase II / regulation of proteolysis / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / histone H4K5ac reader activity / cullin family protein binding / histone H4K12ac reader activity / 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 / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / positive regulation of G2/M transition of mitotic cell cycle / DNA replication initiation / protein monoubiquitination / anatomical structure morphogenesis / negative regulation of signal transduction / positive regulation of T-helper 17 cell lineage commitment / Tat-mediated elongation of the HIV-1 transcript / site of DNA damage / Formation of HIV-1 elongation complex containing HIV-1 Tat / ubiquitin-like ligase-substrate adaptor activity / Formation of HIV elongation complex in the absence of HIV Tat / protein K48-linked ubiquitination / energy homeostasis / RNA Polymerase II Transcription Elongation / neuron projection morphogenesis / cellular response to interferon-beta / Formation of RNA Pol II elongation complex / signal transduction in response to DNA damage / Nuclear events stimulated by ALK signaling in cancer / regulation of proteasomal protein catabolic process / negative regulation of TORC1 signaling / transcription-coupled nucleotide-excision repair / Maturation of protein E / Maturation of protein E / RNA Polymerase II Pre-transcription Events / positive regulation of TORC1 signaling / RNA polymerase II CTD heptapeptide repeat kinase activity / regulation of cellular response to insulin stimulus / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants
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 / Zinc finger, RING-H2-type ...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 / : / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin, N-terminal / Cullin alpha solenoid domain / Cullin / 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 / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Bromodomain, conserved site / Bromodomain signature. / 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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