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- PDB-7mex: Structure of yeast Ubr1 in complex with Ubc2 and N-degron -

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Basic information

Entry
Database: PDB / ID: 7mex
TitleStructure of yeast Ubr1 in complex with Ubc2 and N-degron
Components
  • E3 ubiquitin-protein ligase UBR1
  • N-degron
  • Ubiquitin-conjugating enzyme E2 2
  • Ubiquitin
KeywordsTRANSFERASE / Ubiquitin E3 ligase / ubiquitination / Ubr1 / Ubc2 / Degron / N-end rule
Function / homology
Function and homology information


MUB1-RAD6-UBR2 ubiquitin ligase complex / RAD6-UBR2 ubiquitin ligase complex / Rad6-Rad18 complex / regulation of dipeptide transport / UBR1-RAD6 ubiquitin ligase complex / error-free postreplication DNA repair / HULC complex / proteasome regulatory particle binding / stress-induced homeostatically regulated protein degradation pathway / ubiquitin-dependent protein catabolic process via the N-end rule pathway ...MUB1-RAD6-UBR2 ubiquitin ligase complex / RAD6-UBR2 ubiquitin ligase complex / Rad6-Rad18 complex / regulation of dipeptide transport / UBR1-RAD6 ubiquitin ligase complex / error-free postreplication DNA repair / HULC complex / proteasome regulatory particle binding / stress-induced homeostatically regulated protein degradation pathway / ubiquitin-dependent protein catabolic process via the N-end rule pathway / meiotic DNA double-strand break formation / cytoplasm protein quality control by the ubiquitin-proteasome system / mitochondria-associated ubiquitin-dependent protein catabolic process / Antigen processing: Ubiquitination & Proteasome degradation / telomere maintenance via recombination / proteasome regulatory particle, base subcomplex / ribosome-associated ubiquitin-dependent protein catabolic process / E2 ubiquitin-conjugating enzyme / DNA duplex unwinding / error-free translesion synthesis / sporulation resulting in formation of a cellular spore / proteasome binding / ubiquitin conjugating enzyme activity / protein monoubiquitination / subtelomeric heterochromatin formation / error-prone translesion synthesis / ubiquitin ligase complex / : / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / Endosomal Sorting Complex Required For Transport (ESCRT) / NOTCH2 Activation and Transmission of Signal to the Nucleus / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / Regulation of FZD by ubiquitination / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / mitotic G1 DNA damage checkpoint signaling / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / TCF dependent signaling in response to WNT / SCF-beta-TrCP mediated degradation of Emi1 / Regulation of NF-kappa B signaling / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / Negative regulators of DDX58/IFIH1 signaling / TNFR2 non-canonical NF-kB pathway / NOTCH3 Activation and Transmission of Signal to the Nucleus / activated TAK1 mediates p38 MAPK activation / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / Deactivation of the beta-catenin transactivating complex
Similarity search - Function
E3 ubiquitin-protein ligase UBR-like, C-terminal / Proteolysis_6 C-terminal / E3 ubiquitin-protein ligase UBR1-like / Putative zinc finger in N-recognin (UBR box) / Zinc finger, UBR-type / Zinc finger UBR-type profile. / Putative zinc finger in N-recognin, a recognition component of the N-end rule pathway / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues ...E3 ubiquitin-protein ligase UBR-like, C-terminal / Proteolysis_6 C-terminal / E3 ubiquitin-protein ligase UBR1-like / Putative zinc finger in N-recognin (UBR box) / Zinc finger, UBR-type / Zinc finger UBR-type profile. / Putative zinc finger in N-recognin, a recognition component of the N-end rule pathway / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin-conjugating enzyme E2 2 / Polyubiquitin-C / E3 ubiquitin-protein ligase UBR1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Homo sapiens (human)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsPan, M. / Zheng, Q. / Wang, T. / Liang, L. / Yu, Y. / Liu, L. / Zhao, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2021
Title: Structural insights into Ubr1-mediated N-degron polyubiquitination.
Authors: Man Pan / Qingyun Zheng / Tian Wang / Lujun Liang / Junxiong Mao / Chong Zuo / Ruichao Ding / Huasong Ai / Yuan Xie / Dong Si / Yuanyuan Yu / Lei Liu / Minglei Zhao /
Abstract: The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N- ...The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.
History
DepositionApr 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 22, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
C: Ubiquitin
B: Ubiquitin-conjugating enzyme E2 2
D: N-degron
A: E3 ubiquitin-protein ligase UBR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,95811
Polymers255,5004
Non-polymers4587
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ubiquitin /


Mass: 8622.922 Da / Num. of mol.: 1 / Mutation: G76C / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P0CG48
#2: Protein Ubiquitin-conjugating enzyme E2 2 / E2 ubiquitin-conjugating enzyme 2 / Radiation sensitivity protein 6 / Ubiquitin carrier protein ...E2 ubiquitin-conjugating enzyme 2 / Radiation sensitivity protein 6 / Ubiquitin carrier protein UBC2 / Ubiquitin-protein ligase UBC2


Mass: 17203.389 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RAD6, UBC2, YGL058W / Plasmid: p416CYC / Production host: Saccharomyces cerevisiae S288C (yeast)
References: UniProt: P06104, E2 ubiquitin-conjugating enzyme
#3: Protein/peptide N-degron


Mass: 4571.223 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#4: Protein E3 ubiquitin-protein ligase UBR1 / N-end-recognizing protein / N-recognin-1 / RING-type E3 ubiquitin transferase UBR1


Mass: 225102.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: UBR1, PTR1, YGR184C, G7168 / Plasmid: p416CYC / Production host: Saccharomyces cerevisiae S288C (yeast)
References: UniProt: P19812, RING-type E3 ubiquitin transferase
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: yeast Ubr1 in complex with Ubc2 and N-degron / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 232915 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216279
ELECTRON MICROSCOPYf_angle_d0.48422026
ELECTRON MICROSCOPYf_dihedral_angle_d3.9572126
ELECTRON MICROSCOPYf_chiral_restr0.0392468
ELECTRON MICROSCOPYf_plane_restr0.0032818

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