+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-18056 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | HACE1 in complex with RAC1 Q61L | |||||||||
Map data | ||||||||||
Sample |
| |||||||||
Keywords | E3 / 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 / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / engulfment of apoptotic cell / 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 / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / engulfment of apoptotic cell / NTRK2 activates RAC1 / Inactivation of CDC42 and RAC1 / NADPH oxidase complex / respiratory burst / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / cortical cytoskeleton organization / hepatocyte growth factor receptor signaling pathway / HECT-type E3 ubiquitin transferase / ruffle organization / cell projection assembly / thioesterase binding / regulation of stress fiber assembly / negative regulation of fibroblast migration / RHO GTPases activate CIT / sphingosine-1-phosphate receptor signaling pathway / Nef and signal transduction / motor neuron axon guidance / PCP/CE pathway / positive regulation of neutrophil chemotaxis / regulation of nitric oxide biosynthetic process / RHO GTPases activate KTN1 / Activation of RAC1 / 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 / positive regulation of Rho protein signal transduction / regulation of cell size / establishment or maintenance of cell polarity / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / Golgi cisterna membrane / 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 / Sema3A PAK dependent Axon repulsion / ficolin-1-rich granule membrane / RHO GTPases Activate NADPH Oxidases / EPH-ephrin mediated repulsion of cells / 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 / positive regulation of substrate adhesion-dependent cell spreading / RHO GTPases activate PKNs / Signal transduction by L1 / positive regulation of stress fiber assembly / GPVI-mediated activation cascade / positive regulation of microtubule polymerization / EPHB-mediated forward signaling / RAC1 GTPase cycle / regulation of cell migration / actin filament polymerization / positive regulation of endothelial cell migration / substrate adhesion-dependent cell spreading / cell chemotaxis / cell-matrix adhesion / small monomeric GTPase / secretory granule membrane / VEGFR2 mediated vascular permeability / G protein activity / actin filament organization / Translocation of SLC2A4 (GLUT4) to the plasma membrane / cell motility / RHO GTPases Activate Formins / regulation of actin cytoskeleton organization / FCERI mediated MAPK activation / FCGR3A-mediated phagocytosis / ruffle membrane / neuron migration / MAPK6/MAPK4 signaling / trans-Golgi network / Signaling by SCF-KIT / Regulation of actin dynamics for phagocytic cup formation / Constitutive Signaling by Aberrant PI3K in Cancer / small GTPase binding / VEGFA-VEGFR2 Pathway / response to wounding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
Authors | Wolter M / Duering J / Dienemann C / Lorenz S | |||||||||
Funding support | European Union, Germany, 2 items
| |||||||||
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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_18056.map.gz | 3.9 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-18056-v30.xml emd-18056.xml | 18.2 KB 18.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_18056_fsc.xml | 11.1 KB | Display | FSC data file |
Images | emd_18056.png | 79 KB | ||
Filedesc metadata | emd-18056.cif.gz | 6.5 KB | ||
Others | emd_18056_half_map_1.map.gz emd_18056_half_map_2.map.gz | 95.5 MB 95.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-18056 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-18056 | HTTPS FTP |
-Related structure data
Related structure data | 8q0nMC 8pwlC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_18056.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Voxel size | X=Y=Z: 0.834 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Half map: #2
File | emd_18056_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #1
File | emd_18056_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : HACE1 in complex with RAC1 Q61L
Entire | Name: HACE1 in complex with RAC1 Q61L |
---|---|
Components |
|
-Supramolecule #1: HACE1 in complex with RAC1 Q61L
Supramolecule | Name: HACE1 in complex with RAC1 Q61L / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
---|---|
Source (natural) | Organism: Homo sapiens (human) |
-Macromolecule #1: E3 ubiquitin-protein ligase HACE1
Macromolecule | Name: 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 weight | Theoretical: 99.930656 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: Ras-related C3 botulinum toxin substrate 1
Macromolecule | Name: 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 weight | Theoretical: 23.769672 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: 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 |
-Macromolecule #3: iodoacetic acid
Macromolecule | Name: iodoacetic acid / type: ligand / ID: 3 / Number of copies: 1 / Formula: 04E |
---|---|
Molecular weight | Theoretical: 185.948 Da |
Chemical component information | ChemComp-04E: |
-Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP |
---|---|
Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ChemComp-GTP: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.6 mg/mL | ||||||||
---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 8 Component:
| ||||||||
Grid | Model: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
---|---|
Refinement | Protocol: RIGID BODY FIT |
Output model | PDB-8q0n: |