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- EMDB-18056: HACE1 in complex with RAC1 Q61L -

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Basic information

Entry
Database: EMDB / ID: EMD-18056
TitleHACE1 in complex with RAC1 Q61L
Map data
Sample
  • Complex: HACE1 in complex with RAC1 Q61L
    • Protein or peptide: E3 ubiquitin-protein ligase HACE1
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: iodoacetic acid
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
KeywordsE3 / ubiquitin ligase / small GTPase / crosslink / SIA / LIGASE
Function / homology
Function and homology information


regulation of respiratory burst / negative regulation of interleukin-23 production / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 ...regulation of respiratory burst / negative regulation of interleukin-23 production / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / engulfment of apoptotic cell / Inactivation of CDC42 and RAC1 / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / cortical cytoskeleton organization / respiratory burst / hepatocyte growth factor receptor signaling pathway / HECT-type E3 ubiquitin transferase / ruffle organization / cell projection assembly / thioesterase binding / negative regulation of fibroblast migration / regulation of stress fiber assembly / RHO GTPases activate CIT / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / PCP/CE pathway / motor neuron axon guidance / RHO GTPases activate KTN1 / regulation of nitric oxide biosynthetic process / regulation of lamellipodium assembly / positive regulation of neutrophil chemotaxis / Azathioprine ADME / Activation of RAC1 / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / MET activates RAP1 and RAC1 / Wnt signaling pathway, planar cell polarity pathway / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / lamellipodium assembly / positive regulation of Rho protein signal transduction / Golgi cisterna membrane / establishment or maintenance of cell polarity / regulation of cell size / DSCAM interactions / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / Rho GDP-dissociation inhibitor binding / NRAGE signals death through JNK / Rac protein signal transduction / Golgi organization / RHO GTPases activate PAKs / positive regulation of focal adhesion assembly / semaphorin-plexin signaling pathway / ficolin-1-rich granule membrane / Sema3A PAK dependent Axon repulsion / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate NADPH Oxidases / RHO GTPases Activate WASPs and WAVEs / anatomical structure morphogenesis / protein K48-linked ubiquitination / RHO GTPases activate IQGAPs / localization / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / regulation of cell migration / positive regulation of microtubule polymerization / RHO GTPases activate PKNs / positive regulation of stress fiber assembly / GPVI-mediated activation cascade / RAC1 GTPase cycle / EPHB-mediated forward signaling / actin filament polymerization / cell chemotaxis / substrate adhesion-dependent cell spreading / cell-matrix adhesion / small monomeric GTPase / G protein activity / positive regulation of endothelial cell migration / secretory granule membrane / VEGFR2 mediated vascular permeability / Signal transduction by L1 / actin filament organization / cell motility / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of actin cytoskeleton organization / FCGR3A-mediated phagocytosis / FCERI mediated MAPK activation / neuron migration / MAPK6/MAPK4 signaling / trans-Golgi network / Signaling by SCF-KIT / cytoplasmic ribonucleoprotein granule / Regulation of actin dynamics for phagocytic cup formation / ruffle membrane / small GTPase binding
Similarity search - Function
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. / Ankyrin repeats (many copies) / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases ...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. / Ankyrin repeats (many copies) / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ras-related C3 botulinum toxin substrate 1 / E3 ubiquitin-protein ligase HACE1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWolter M / Duering J / Dienemann C / Lorenz S
Funding supportEuropean Union, Germany, 2 items
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, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18056.png

Downloads & links

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Map

FileDownload / File: emd_18056.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-6.2318587 - 8.421029000000001
Average (Standard dev.)0.008510813 (±0.123904176)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18056_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18056_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : HACE1 in complex with RAC1 Q61L

EntireName: HACE1 in complex with RAC1 Q61L
Components
  • Complex: HACE1 in complex with RAC1 Q61L
    • Protein or peptide: E3 ubiquitin-protein ligase HACE1
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: iodoacetic acid
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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Supramolecule #1: HACE1 in complex with RAC1 Q61L

SupramoleculeName: HACE1 in complex with RAC1 Q61L / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: E3 ubiquitin-protein ligase HACE1

MacromoleculeName: E3 ubiquitin-protein ligase HACE1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.930656 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GELPEDNETA VYTLMPMVMA DQHRSVSELL SNSKFDVNYA FGRVKRSLLH IAANCGSVEC LVLLLKKGAN PNYQDISGCT PLHLAARNG QKKCMSKLLE YSADVNICNN EGLTAIHWLA VNGRTELLHD LVQHVSDVDV EDAMGQTALH VACQNGHKTT V QCLLDSGA ...String:
GELPEDNETA VYTLMPMVMA DQHRSVSELL SNSKFDVNYA FGRVKRSLLH IAANCGSVEC LVLLLKKGAN PNYQDISGCT PLHLAARNG QKKCMSKLLE YSADVNICNN EGLTAIHWLA VNGRTELLHD LVQHVSDVDV EDAMGQTALH VACQNGHKTT V QCLLDSGA DINRPNVSGA TPLYFACSHG QRDTAQILLL RGAKYLPDKN GVTPLDLCVQ GGYGETCEVL IQYHPRLFQT II QMTQNED LRENMLRQVL EHLSQQSESQ YLKILTSLAE VATTNGHKLL SLSSNYDAQM KSLLRIVRMF CHVFRIGPSS PSN GIDMGY NGNKTPRSQV FKPLELLWHS LDEWLVLIAT ELMKNKRDST EITSILLKQK GQDQDAASIP PFEPPGPGSY ENLS TGTRE SKPDALAGRQ EASADCQDVI SMTANRLSAV IQAFYMCCSC QMPPGMTSPR FIEFVCKHDE VLKCFVNRNP KIIFD HFHF LLECPELMSR FMHIIKAQPF KDRCEWFYEH LHSGQPDSDM VHRPVNENDI LLVHRDSIFR SSCEVVSKAN CAKLKQ GIA VRFHGEEGMG QGVVREWFDI LSNEIVNPDY ALFTQSADGT TFQPNSNSYV NPDHLNYFRF AGQILGLALN HRQLVNI YF TRSFYKHILG IPVNYQDVAS IDPEYAKNLQ WILDNDISDL GLELTFSVET DVFGAMEEVP LKPGGGSILV TQNNKAEY V QLVTELRMTR AIQPQINAFL QGFHMFIPPS LIQLFDEYEL ELLLSGMPEI DVSDWIKNTE YTSGYEREDP VIQWFWEVV EDITQEERVL LLQFVTGSSR VPHGGFANIM GGSGLQNFTI AAVPYTPNLL PTSSTCINML KLPEYPSKEI LKDRLLVALH CGSYGYTMA

UniProtKB: E3 ubiquitin-protein ligase HACE1

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Macromolecule #2: Ras-related C3 botulinum toxin substrate 1

MacromoleculeName: Ras-related C3 botulinum toxin substrate 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.769672 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH SSGLEVLFQG PMQAIKCVVV GDGAVGKTCL LISYTTNAFP GEYIPTVFDN YSANVMVDGK PVNLGLWDTA GLEDYDRLR PLSYPQTDVF LICFSLVSPA SFENVRAKWY PEVRHHCPNT PIILVGTKLD LRDDKDTIEK LKEKKLTPIT Y PQGLAMAK ...String:
MGSSHHHHHH SSGLEVLFQG PMQAIKCVVV GDGAVGKTCL LISYTTNAFP GEYIPTVFDN YSANVMVDGK PVNLGLWDTA GLEDYDRLR PLSYPQTDVF LICFSLVSPA SFENVRAKWY PEVRHHCPNT PIILVGTKLD LRDDKDTIEK LKEKKLTPIT Y PQGLAMAK EIGAVKYLEC SALTQRGLKT VFDEAIRAVL CPPPVKKRKR KCLLL

UniProtKB: Ras-related C3 botulinum toxin substrate 1

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Macromolecule #3: iodoacetic acid

MacromoleculeName: iodoacetic acid / type: ligand / ID: 3 / Number of copies: 1 / Formula: 04E
Molecular weightTheoretical: 185.948 Da
Chemical component information

ChemComp-04E:
iodoacetic acid / Iodoacetic acid

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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

Concentration0.6 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
20.0 mMHEPES
50.0 mMNaClSodium chloride
3.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 256595
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8q0n:
HACE1 in complex with RAC1 Q61L

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