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- PDB-8pwl: Cryo-EM structure of a full-length HACE1 dimer -

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Basic information

Entry
Database: PDB / ID: 8pwl
TitleCryo-EM structure of a full-length HACE1 dimer
ComponentsE3 ubiquitin-protein ligase HACE1
KeywordsLIGASE / E3 / HECT / ubiquitin
Function / homology
Function and homology information


HECT-type E3 ubiquitin transferase / Golgi cisterna membrane / Golgi organization / Rac protein signal transduction / protein K48-linked ubiquitination / regulation of cell migration / small GTPase binding / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation ...HECT-type E3 ubiquitin transferase / Golgi cisterna membrane / Golgi organization / Rac protein signal transduction / protein K48-linked ubiquitination / regulation of cell migration / small GTPase binding / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / membrane fusion / protein ubiquitination / nuclear body / Golgi membrane / endoplasmic reticulum / nucleus / cytoplasm
Similarity search - Function
: / Ankyrin repeats (many copies) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. ...: / Ankyrin repeats (many copies) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase HACE1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.73 Å
AuthorsDuering, J. / Wolter, M. / Dienemann, C. / Lorenz, S.
Funding support Germany, European Union, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
European Molecular Biology Organization (EMBO)EMBO ALTF 439-2022European Union
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1.
Authors: Jonas Düring / Madita Wolter / Julia J Toplak / Camilo Torres / Olexandr Dybkov / Thornton J Fokkens / Katherine E Bohnsack / Henning Urlaub / Wieland Steinchen / Christian Dienemann / Sonja Lorenz /
Abstract: Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of ...Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.
History
DepositionJul 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Feb 28, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase HACE1
B: E3 ubiquitin-protein ligase HACE1


Theoretical massNumber of molelcules
Total (without water)205,0132
Polymers205,0132
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein E3 ubiquitin-protein ligase HACE1 / HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 / HECT-type E3 ubiquitin ...HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 / HECT-type E3 ubiquitin transferase HACE1


Mass: 102506.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HACE1, KIAA1320 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: Q8IYU2, HECT-type E3 ubiquitin transferase

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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: HACE1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
230 mMNaCl1
31 mMDTT1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 29748

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Processing

EM software
IDNameVersionCategory
1Warp1.0.9particle selection
2SerialEMimage acquisition
4Warp1.0.9CTF correction
7PHENIX1.20.1model fitting
9PHENIX1.20.1model refinement
10ISOLDE1.6.1model refinement
11cryoSPARCv4initial Euler assignment
12cryoSPARCv4final Euler assignment
13cryoSPARCv4classification
14cryoSPARCv43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3639240
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118791 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212527
ELECTRON MICROSCOPYf_angle_d0.50316975
ELECTRON MICROSCOPYf_dihedral_angle_d3.611659
ELECTRON MICROSCOPYf_chiral_restr0.0371905
ELECTRON MICROSCOPYf_plane_restr0.0032206

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