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- PDB-8q0n: HACE1 in complex with RAC1 Q61L -

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Basic information

Entry
Database: PDB / ID: 8q0n
TitleHACE1 in complex with RAC1 Q61L
Components
  • E3 ubiquitin-protein ligase HACE1
  • Ras-related C3 botulinum toxin substrate 1
KeywordsLIGASE / E3 / ubiquitin ligase / small GTPase / crosslink / SIA
Function / homology
Function and homology information


regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex ...regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / respiratory burst / WNT5:FZD7-mediated leishmania damping / cortical cytoskeleton organization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / hepatocyte growth factor receptor signaling pathway / ruffle organization / cell projection assembly / positive regulation of bicellular tight junction assembly / thioesterase binding / HECT-type E3 ubiquitin transferase / regulation of stress fiber assembly / regulation of lamellipodium assembly / negative regulation of fibroblast migration / RHO GTPases activate CIT / regulation of nitric oxide biosynthetic process / motor neuron axon guidance / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / PCP/CE pathway / Activation of RAC1 / RHO GTPases activate KTN1 / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / Azathioprine ADME / positive regulation of cell-substrate adhesion / positive regulation of neutrophil chemotaxis / Sema4D mediated inhibition of cell attachment and migration / Ephrin signaling / CD28 dependent Vav1 pathway / superoxide anion generation / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / Golgi cisterna membrane / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / NRAGE signals death through JNK / regulation of cell size / positive regulation of Rho protein signal transduction / Rho GDP-dissociation inhibitor binding / establishment or maintenance of cell polarity / Golgi organization / Rac protein signal transduction / RHO GTPases activate PAKs / semaphorin-plexin signaling pathway / ficolin-1-rich granule membrane / Sema3A PAK dependent Axon repulsion / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / positive regulation of focal adhesion assembly / anatomical structure morphogenesis / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / protein K48-linked ubiquitination / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / RHO GTPases activate PKNs / GPVI-mediated activation cascade / positive regulation of stress fiber assembly / positive regulation of microtubule polymerization / actin filament polymerization / EPHB-mediated forward signaling / RAC1 GTPase cycle / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of endothelial cell migration / substrate adhesion-dependent cell spreading / regulation of cell migration / secretory granule membrane / small monomeric GTPase / actin filament organization / cell-matrix adhesion / Signal transduction by L1 / VEGFR2 mediated vascular permeability / cell projection / Translocation of SLC2A4 (GLUT4) to the plasma membrane / cell chemotaxis / regulation of actin cytoskeleton organization / FCGR3A-mediated phagocytosis / FCERI mediated MAPK activation / cell motility / RHO GTPases Activate Formins / trans-Golgi network / Signaling by SCF-KIT / MAPK6/MAPK4 signaling / small GTPase binding / Regulation of actin dynamics for phagocytic cup formation / response to wounding / VEGFA-VEGFR2 Pathway / cytoplasmic ribonucleoprotein granule / ruffle membrane
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 / Small GTPase Rho / small GTPase Rho family profile. / Rho (Ras homology) subfamily of Ras-like small GTPases ...: / 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 / Small GTPase Rho / small GTPase Rho family profile. / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Rab subfamily of small GTPases / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
iodoacetic acid / GUANOSINE-5'-TRIPHOSPHATE / Ras-related C3 botulinum toxin substrate 1 / E3 ubiquitin-protein ligase HACE1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWolter, M. / Duering, J. / Dienemann, C. / Lorenz, S.
Funding supportEuropean Union, Germany, 2items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)EMBO ALTF 439-2022European Union
Max Planck Society Germany
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 28, 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
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: E3 ubiquitin-protein ligase HACE1
C: Ras-related C3 botulinum toxin substrate 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,4094
Polymers123,7002
Non-polymers7092
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 99930.656 Da / Num. of mol.: 1 / Mutation: deletion 1-21
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
#2: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 23769.672 Da / Num. of mol.: 1 / Mutation: Q61L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P63000, small monomeric GTPase
#3: Chemical ChemComp-04E / iodoacetic acid


Mass: 185.948 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3IO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY

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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 in complex with RAC1 Q61L / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
250 mMNaCl1
33 mMDTT1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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: 3500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1Warpparticle selection
2SerialEMimage acquisition
4WarpCTF correction
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
13ISOLDEmodel refinement
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256595 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028182
ELECTRON MICROSCOPYf_angle_d0.46511113
ELECTRON MICROSCOPYf_dihedral_angle_d7.1871094
ELECTRON MICROSCOPYf_chiral_restr0.0361257
ELECTRON MICROSCOPYf_plane_restr0.0041431

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