+Open data
-Basic information
Entry | Database: PDB / ID: 8q0n | |||||||||
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Title | HACE1 in complex with RAC1 Q61L | |||||||||
Components |
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Keywords | LIGASE / E3 / ubiquitin ligase / small GTPase / crosslink / SIA | |||||||||
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 / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / Inactivation of CDC42 and RAC1 ...regulation of respiratory burst / negative regulation of interleukin-23 production / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / Inactivation of CDC42 and RAC1 / NADPH oxidase complex / engulfment of apoptotic cell / WNT5:FZD7-mediated leishmania damping / respiratory burst / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / cortical cytoskeleton organization / hepatocyte growth factor receptor signaling pathway / HECT-type E3 ubiquitin transferase / ruffle organization / thioesterase binding / regulation of stress fiber assembly / negative regulation of fibroblast migration / cell projection assembly / RHO GTPases activate CIT / sphingosine-1-phosphate receptor signaling pathway / Nef and signal transduction / PCP/CE pathway / positive regulation of neutrophil chemotaxis / regulation of nitric oxide biosynthetic process / RHO GTPases activate KTN1 / Activation of RAC1 / motor neuron axon guidance / regulation of lamellipodium assembly / Azathioprine ADME / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / Wnt signaling pathway, planar cell polarity pathway / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / lamellipodium assembly / regulation of cell size / establishment or maintenance of cell polarity / Golgi cisterna membrane / DSCAM interactions / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / Rho GDP-dissociation inhibitor binding / positive regulation of Rho protein signal transduction / 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 / Sema3A PAK dependent Axon repulsion / ficolin-1-rich granule membrane / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / localization / RHO GTPases activate IQGAPs / anatomical structure morphogenesis / protein K48-linked ubiquitination / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / RHO GTPases activate PKNs / regulation of cell migration / positive regulation of microtubule polymerization / positive regulation of stress fiber assembly / GPVI-mediated activation cascade / EPHB-mediated forward signaling / RAC1 GTPase cycle / actin filament polymerization / positive regulation of endothelial cell migration / substrate adhesion-dependent cell spreading / cell-matrix adhesion / cell chemotaxis / small monomeric GTPase / secretory granule membrane / VEGFR2 mediated vascular permeability / G protein activity / Signal transduction by L1 / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / cell motility / regulation of actin cytoskeleton organization / actin filament organization / FCERI mediated MAPK activation / FCGR3A-mediated phagocytosis / neuron migration / MAPK6/MAPK4 signaling / trans-Golgi network / Signaling by SCF-KIT / Regulation of actin dynamics for phagocytic cup formation / small GTPase binding / ruffle membrane / VEGFA-VEGFR2 Pathway Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
Authors | Wolter, M. / Duering, J. / Dienemann, C. / Lorenz, S. | |||||||||
Funding support | European Union, Germany, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8q0n.cif.gz | 343.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8q0n.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8q0n.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8q0n_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 8q0n_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 8q0n_validation.xml.gz | 43.3 KB | Display | |
Data in CIF | 8q0n_validation.cif.gz | 62.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/q0/8q0n ftp://data.pdbj.org/pub/pdb/validation_reports/q0/8q0n | HTTPS FTP |
-Related structure data
Related structure data | 18056MC 8pwlC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 |
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#2: Protein | 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 / |
#4: Chemical | ChemComp-GTP / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: HACE1 in complex with RAC1 Q61L / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | ||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||
Specimen support | Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256595 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | |||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | |||||||||||||||||||||||||||||||||
Refine LS restraints |
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