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- PDB-7mop: Cryo-EM structure of human HUWE1 in complex with DDIT4 -

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Basic information

Entry
Database: PDB / ID: 7mop
TitleCryo-EM structure of human HUWE1 in complex with DDIT4
Components
  • DNA damage-inducible transcript 4 protein
  • E3 ubiquitin-protein ligase HUWE1
KeywordsTRANSFERASE/Apoptosis / Ubiquitin / Quality Control / E3 ligase / protein degradation / TRANSFERASE / TRANSFERASE-Apoptosis complex
Function / homology
Function and homology information


protein-containing complex disassembly / negative regulation of mitochondrial fusion / histone ubiquitin ligase activity / positive regulation of mitophagy in response to mitochondrial depolarization / HECT-type E3 ubiquitin transferase / negative regulation of glycolytic process / negative regulation of TOR signaling / positive regulation of protein targeting to mitochondrion / protein monoubiquitination / Golgi organization ...protein-containing complex disassembly / negative regulation of mitochondrial fusion / histone ubiquitin ligase activity / positive regulation of mitophagy in response to mitochondrial depolarization / HECT-type E3 ubiquitin transferase / negative regulation of glycolytic process / negative regulation of TOR signaling / positive regulation of protein targeting to mitochondrion / protein monoubiquitination / Golgi organization / neurotrophin TRK receptor signaling pathway / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / 14-3-3 protein binding / reactive oxygen species metabolic process / cellular response to dexamethasone stimulus / positive regulation of protein ubiquitination / TP53 Regulates Metabolic Genes / neuron migration / circadian regulation of gene expression / neuron differentiation / base-excision repair / brain development / protein polyubiquitination / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / secretory granule lumen / defense response to virus / ficolin-1-rich granule lumen / membrane fusion / cell differentiation / response to hypoxia / intracellular signal transduction / Golgi membrane / apoptotic process / Neutrophil degranulation / mitochondrion / DNA binding / RNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / nucleus / cytoplasm / cytosol
Similarity search - Function
RTP801-like / RTP801-like, C-terminal domain superfamily / RTP801 C-terminal region / HUWE1, UBA domain / E3 ubiquitin ligase, domain of unknown function DUF908 / E3 ubiquitin ligase, domain of unknown function DUF913 / Domain of Unknown Function (DUF908) / Domain of Unknown Function (DUF913) / HUWE1/Rev1, ubiquitin binding region / Ubiquitin binding region ...RTP801-like / RTP801-like, C-terminal domain superfamily / RTP801 C-terminal region / HUWE1, UBA domain / E3 ubiquitin ligase, domain of unknown function DUF908 / E3 ubiquitin ligase, domain of unknown function DUF913 / Domain of Unknown Function (DUF908) / Domain of Unknown Function (DUF913) / HUWE1/Rev1, ubiquitin binding region / Ubiquitin binding region / WWE domain / WWE domain superfamily / WWE domain / WWE domain profile. / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / UBA/TS-N domain / Ubiquitin associated domain / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily / Armadillo-type fold
Similarity search - Domain/homology
E3 ubiquitin-protein ligase HUWE1 / DNA damage-inducible transcript 4 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHunkeler, M. / Fischer, E.S.
Funding support United States, Switzerland, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA2144608 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA218278 United States
Swiss National Science Foundation174331 Switzerland
Swiss National Science Foundation191053 Switzerland
Other privateDRR-50-18 United States
CitationJournal: Mol Cell / Year: 2021
Title: Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition.
Authors: Moritz Hunkeler / Cyrus Y Jin / Michelle W Ma / Julie K Monda / Daan Overwijn / Eric J Bennett / Eric S Fischer /
Abstract: HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival ...HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.
History
DepositionMay 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Sep 15, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase HUWE1
B: DNA damage-inducible transcript 4 protein


Theoretical massNumber of molelcules
Total (without water)514,8462
Polymers514,8462
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, elution volume fits with mass standards, mass spectrometry, from pull downs & purified protein, microscopy, Atomic model was built into the maps
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein E3 ubiquitin-protein ligase HUWE1 / ARF-binding protein 1 / ARF-BP1 / HECT / UBA and WWE domain-containing protein 1 / HECT-type E3 ...ARF-binding protein 1 / ARF-BP1 / HECT / UBA and WWE domain-containing protein 1 / HECT-type E3 ubiquitin transferase HUWE1 / Homologous to E6AP carboxyl terminus homologous protein 9 / HectH9 / Large structure of UREB1 / LASU1 / Mcl-1 ubiquitin ligase E3 / Mule / Upstream regulatory element-binding protein 1 / URE-binding protein 1


Mass: 486409.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HUWE1, KIAA0312, KIAA1578, UREB1, HSPC272 / Plasmid: pDEST / Cell line (production host): Expi293 / Production host: Homo sapiens (human)
References: UniProt: Q7Z6Z7, HECT-type E3 ubiquitin transferase
#2: Protein DNA damage-inducible transcript 4 protein / HIF-1 responsive protein RTP801 / Protein regulated in development and DNA damage response 1 / REDD-1


Mass: 28436.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDIT4, REDD1, RTP801 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NX09

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1E3 ubiquitin-protein ligase HUWE1 in complex with DDIT4ORGANELLE OR CELLULAR COMPONENTfull lengthall0RECOMBINANT
2E3 ubiquitin-protein ligase HUWE1ORGANELLE OR CELLULAR COMPONENTfull length#11RECOMBINANT
3DNA damage-inducible transcript 4 proteinORGANELLE OR CELLULAR COMPONENTfull length#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.5 MDaNO
12
210.028 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Homo sapiens (human)9606Expi293
32Homo sapiens (human)9606Expi293
43Trichoplusia ni (cabbage looper)7111pAC8
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)C8H18N2O4S1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K
Details: CHAPSO detergent added to final conc. of 1 mM. Sample applied twice.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Data collection in counting mode, using multi-shot scheme (4 holes per stage position, 2 movies per hole)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.497 sec. / Electron dose: 53.34 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7208
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
5RELIONCTF correction
8Cootmodel fitting
9PHENIXmodel fitting
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
13RELIONclassification
14RELION3D reconstruction
15PHENIXmodel refinement
CTF correctionDetails: standard correction in Relion / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1673531
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 312798 / Algorithm: BACK PROJECTION / Details: as implemented in relion / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 74 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CC
Atomic model buildingPDB-ID: 7JQ9

7jq9


Pdb chain-ID: A / Accession code: 7JQ9 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005619715
ELECTRON MICROSCOPYf_angle_d0.793326687
ELECTRON MICROSCOPYf_chiral_restr0.04553154
ELECTRON MICROSCOPYf_plane_restr0.00723361
ELECTRON MICROSCOPYf_dihedral_angle_d17.67082618

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