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Open data
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Basic information
Entry | Database: PDB / ID: 8q0n | |||||||||
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Title | HACE1 in complex with RAC1 Q61L | |||||||||
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![]() | LIGASE / E3 / ubiquitin ligase / small GTPase / crosslink / SIA | |||||||||
Function / homology | ![]() embryonic olfactory bulb interneuron precursor migration / anatomical structure arrangement / regulation of ERK5 cascade / angiotensin-activated signaling pathway involved in heart process / positive regulation of ovarian follicle development / cerebral cortex GABAergic interneuron development / regulation of respiratory burst / auditory receptor cell morphogenesis / cerebral cortex radially oriented cell migration / erythrocyte enucleation ...embryonic olfactory bulb interneuron precursor migration / anatomical structure arrangement / regulation of ERK5 cascade / angiotensin-activated signaling pathway involved in heart process / positive regulation of ovarian follicle development / cerebral cortex GABAergic interneuron development / regulation of respiratory burst / auditory receptor cell morphogenesis / cerebral cortex radially oriented cell migration / erythrocyte enucleation / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of hydrogen peroxide metabolic process / interneuron migration / kinocilium / regulation of cell adhesion involved in heart morphogenesis / negative regulation of receptor-mediated endocytosis / engulfment of apoptotic cell / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / cochlea morphogenesis / regulation of neuron maturation / respiratory burst / WNT5:FZD7-mediated leishmania damping / cortical cytoskeleton organization / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / positive regulation of skeletal muscle acetylcholine-gated channel clustering / hepatocyte growth factor receptor signaling pathway / GTP-dependent protein binding / midbrain dopaminergic neuron differentiation / ruffle organization / epithelial cell morphogenesis / cell projection assembly / positive regulation of bicellular tight junction assembly / thioesterase binding / HECT-type E3 ubiquitin transferase / regulation of lamellipodium assembly / regulation of stress fiber assembly / regulation of neuron migration / negative regulation of fibroblast migration / RHO GTPases activate CIT / cell-cell junction organization / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / motor neuron axon guidance / regulation of nitric oxide biosynthetic process / PCP/CE pathway / Activation of RAC1 / RHO GTPases activate KTN1 / MET activates RAP1 and RAC1 / positive regulation of neutrophil chemotaxis / DCC mediated attractive signaling / Azathioprine ADME / Sema4D mediated inhibition of cell attachment and migration / hyperosmotic response / positive regulation of cell-substrate adhesion / Ephrin signaling / CD28 dependent Vav1 pathway / positive regulation of ruffle assembly / superoxide anion generation / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / regulation of receptor signaling pathway via JAK-STAT / Golgi cisterna membrane / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / NRAGE signals death through JNK / dendrite morphogenesis / regulation of cell size / Rho GDP-dissociation inhibitor binding / positive regulation of Rho protein signal transduction / establishment or maintenance of cell polarity / synaptic transmission, GABAergic / positive regulation of dendritic spine development / positive regulation of actin filament polymerization / Golgi organization / Rac protein signal transduction / RHO GTPases activate PAKs / pericentriolar material / semaphorin-plexin signaling pathway / ficolin-1-rich granule membrane / Sema3A PAK dependent Axon repulsion / regulation of postsynapse assembly / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / anatomical structure morphogenesis / regulation of neuronal synaptic plasticity / positive regulation of focal adhesion assembly / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / positive regulation of insulin secretion involved in cellular response to glucose stimulus / phagocytic cup / protein K48-linked ubiquitination / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
![]() | Wolter, M. / Duering, J. / Dienemann, C. / Lorenz, S. | |||||||||
Funding support | European Union, ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 343.6 KB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 43.3 KB | Display | |
Data in CIF | ![]() | 62.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 18056MC ![]() 8pwlC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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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.) ![]() ![]() ![]() 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.) ![]() ![]() ![]() |
#3: Chemical | ChemComp-04E / |
#4: Chemical | ChemComp-GTP / |
Has ligand of interest | Y |
Has protein modification | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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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: ![]() | ||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||
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 |
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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: ![]() |
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) |
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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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