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- EMDB-51373: Full-lenght Nedd4-2 E3 ubiquitin ligase -

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Basic information

Entry
Database: EMDB / ID: EMD-51373
TitleFull-lenght Nedd4-2 E3 ubiquitin ligase
Map datamain map, phenix.autosharpen
Sample
  • Complex: Nedd4-2
    • Protein or peptide: Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like
KeywordsNedd4-2 / calcium / 14-3-3 proteins / H/D exchange / Cryo-EM / SAXS / protein-protein interaction / SIGNALING PROTEIN
Function / homology
Function and homology information


negative regulation of sodium ion import across plasma membrane / positive regulation of caveolin-mediated endocytosis / RING-type E3 ubiquitin transferase (cysteine targeting) / negative regulation of sodium ion transmembrane transport / regulation of potassium ion transmembrane transporter activity / negative regulation of potassium ion transmembrane transport / : / negative regulation of potassium ion transmembrane transporter activity / negative regulation of protein localization to cell surface / regulation of membrane repolarization ...negative regulation of sodium ion import across plasma membrane / positive regulation of caveolin-mediated endocytosis / RING-type E3 ubiquitin transferase (cysteine targeting) / negative regulation of sodium ion transmembrane transport / regulation of potassium ion transmembrane transporter activity / negative regulation of potassium ion transmembrane transport / : / negative regulation of potassium ion transmembrane transporter activity / negative regulation of protein localization to cell surface / regulation of membrane repolarization / regulation of sodium ion transmembrane transport / regulation of membrane depolarization / potassium channel inhibitor activity / receptor catabolic process / positive regulation of dendrite extension / ventricular cardiac muscle cell action potential / regulation of monoatomic ion transmembrane transport / HECT-type E3 ubiquitin transferase / sodium channel inhibitor activity / regulation of dendrite morphogenesis / neuromuscular junction development / regulation of synapse organization / sodium channel regulator activity / protein monoubiquitination / potassium channel regulator activity / protein K48-linked ubiquitination / multivesicular body / Downregulation of TGF-beta receptor signaling / regulation of membrane potential / Downregulation of SMAD2/3:SMAD4 transcriptional activity / regulation of protein stability / Budding and maturation of HIV virion / receptor internalization / Stimuli-sensing channels / neuron projection development / positive regulation of protein catabolic process / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / monoatomic ion transmembrane transport / proteasome-mediated ubiquitin-dependent protein catabolic process / transmembrane transporter binding / protein ubiquitination / apical plasma membrane / Golgi apparatus / extracellular exosome / nucleoplasm / cytosol / cytoplasm
Similarity search - Function
E3 ubiquitin-protein ligase, SMURF1 type / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Protein kinase C conserved region 2 (CalB) / C2 domain / WW domain ...E3 ubiquitin-protein ligase, SMURF1 type / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Protein kinase C conserved region 2 (CalB) / C2 domain / WW domain / WW/rsp5/WWP domain signature. / C2 domain / C2 domain profile. / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain / C2 domain superfamily
Similarity search - Domain/homology
E3 ubiquitin-protein ligase NEDD4-like
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsKosek D / Janosev M / Obsil T / Obsilova V
Funding support Czech Republic, 1 items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic23-046865 Czech Republic
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of ubiquitin ligase Nedd4-2 autoinhibition and regulation by calcium and 14-3-3 proteins.
Authors: Masa Janosev / Dalibor Kosek / Andrej Tekel / Rohit Joshi / Karolina Honzejkova / Pavel Pohl / Tomas Obsil / Veronika Obsilova /
Abstract: Nedd4-2 E3 ligase regulates Na homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various ...Nedd4-2 E3 ligase regulates Na homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.
History
DepositionAug 19, 2024-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51373.png

Downloads & links

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Map

FileDownload / File: emd_51373.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map, phenix.autosharpen
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 320 pix.
= 266.752 Å		0.83 Å/pix.
x 320 pix.
= 266.752 Å		0.83 Å/pix.
x 320 pix.
= 266.752 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8336 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-35.757744000000002 - 57.694789999999998
Average (Standard dev.)-0.000000000000373 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.75198 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51373_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: raw map

Fileemd_51373_additional_1.map
Annotationraw map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_51373_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_51373_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Nedd4-2

EntireName: Nedd4-2
Components
  • Complex: Nedd4-2
    • Protein or peptide: Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like

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Supramolecule #1: Nedd4-2

SupramoleculeName: Nedd4-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like

MacromoleculeName: Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like / 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: 110.472945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GLGSMATGLG EPVYGLSEDE GESRILRVKV VSGIDLAKKD IFGASDPYVK LSLYVADENR ELALVQTKTI KKTLNPKWNE EFYFRVNPS NHRLLFEVFD ENRLTRDDFL GQVDVPLSHL PTEDPTMERP YTFKDFLLRP RSHKSRVKGF LRLKMAYMPK N GGQDEENS ...String:
GLGSMATGLG EPVYGLSEDE GESRILRVKV VSGIDLAKKD IFGASDPYVK LSLYVADENR ELALVQTKTI KKTLNPKWNE EFYFRVNPS NHRLLFEVFD ENRLTRDDFL GQVDVPLSHL PTEDPTMERP YTFKDFLLRP RSHKSRVKGF LRLKMAYMPK N GGQDEENS DQRDDMEHGW EVVDSNDSAS QHQEELPPPP LPPGWEEKVD NLGRTYYVNH NNRTTQWHRP SLMDVSSESD NN IRQINQE AAHRRFRSRR HISEDLEPEP SEGGDVPEPW ETISEEVNIA GDSLGLALPP PPASPGSRTS PQELSEELSR RLQ ITPDSN GEQFSSLIQR EPSSRLRSCS VTDAVAEQGH LPPPSVAYVH TTPGLPSGWE ERKDAKGRTY YVNHNNRTTT WTRP IMQLA EDGASGSATN SNNHLIEPQI RRPRSLSSPT VTLSAPLEGA KDSPVRRAVK DTLSNPQSPQ PSPYNSPKPQ HKVTQ SFLP PGWEMRIAPN GRPFFIDHNT KTTTWEDPRL KFPVHMRSKT SLNPNDLGPL PPGWEERIHL DGRTFYIDHN SKITQW EDP RLQNPAITGP AVPYSREFKQ KYDYFRKKLK KPADIPNRFE MKLHRNNIFE ESYRRIMSVK RPDVLKARLW IEFESEK GL DYGGVAREWF FLLSKEMFNP YYGLFEYSAT DNYTLQINPN SGLCNEDHLS YFTFIGRVAG LAVFHGKLLD GFFIRPFY K MMLGKQITLN DMESVDSEYY NSLKWILEND PTELDLMFCI DEENFGQTYQ VDLKPNGSEI MVTNENKREY IDLVIQWRF VNRVQKQMNA FLEGFTELLP IDLIKIFDEN ELELLMCGLG DVDVNDWRQH SIYKNGYCPN HPVIQWFWKA VLLMDAEKRI RLLQFVTGT SRVPMNGFAE LYGSNGPQLF TIEQWGSPEK LPRAHTCFNR LDLPPYETFE DLREKLLMAV ENAQGFEGVD

UniProtKB: E3 ubiquitin-protein ligase NEDD4-like

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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

Concentration2.7 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 130 mM KCl, 20 mM NaCl, 1 mM EDTA, 1 mM TCEP, 3mM CHAPSO
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1.) / Number images used: 225759
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.4.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.4.1.)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

2oni

source_name: PDB, initial_model_type: experimental model

2nsq

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL
Output model

PDB-9gik:
Full-lenght Nedd4-2 E3 ubiquitin ligase

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