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

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Basic information

Entry
Database: PDB / ID: 9gik
TitleFull-lenght Nedd4-2 E3 ubiquitin ligase
ComponentsIsoform 5 of E3 ubiquitin-protein ligase NEDD4-like
KeywordsSIGNALING PROTEIN / Nedd4-2 / calcium / 14-3-3 proteins / H/D exchange / Cryo-EM / SAXS / protein-protein interaction
Function / homology
Function and homology information


positive regulation of caveolin-mediated endocytosis / RING-type E3 ubiquitin transferase (cysteine targeting) / negative regulation of sodium ion transmembrane transport / negative regulation of sodium ion import across plasma membrane / negative regulation of potassium ion export across plasma membrane / negative regulation of potassium ion transmembrane transport / negative regulation of protein localization to cell surface / regulation of membrane repolarization / positive regulation of dendrite extension / regulation of membrane depolarization ...positive regulation of caveolin-mediated endocytosis / RING-type E3 ubiquitin transferase (cysteine targeting) / negative regulation of sodium ion transmembrane transport / negative regulation of sodium ion import across plasma membrane / negative regulation of potassium ion export across plasma membrane / negative regulation of potassium ion transmembrane transport / negative regulation of protein localization to cell surface / regulation of membrane repolarization / positive regulation of dendrite extension / regulation of membrane depolarization / receptor catabolic process / regulation of sodium ion transmembrane transport / potassium channel inhibitor activity / ventricular cardiac muscle cell action potential / 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 / protein K48-linked ubiquitination / multivesicular body / Downregulation of TGF-beta receptor signaling / regulation of membrane potential / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Budding and maturation of HIV virion / regulation of protein stability / receptor internalization / Stimuli-sensing channels / neuron projection development / ubiquitin-protein transferase activity / positive regulation of protein catabolic process / 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 / C2 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 / C2 domain / C2 domain profile. / WW domain / WW/rsp5/WWP domain signature. / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsKosek, D. / Janosev, M. / Obsil, T. / Obsilova, V.
Funding support Czech Republic, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like


Theoretical massNumber of molelcules
Total (without water)110,4731
Polymers110,4731
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like / HECT-type E3 ubiquitin transferase NED4L / NEDD4.2 / Nedd4-2


Mass: 110472.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NEDD4L, KIAA0439, NEDL3 / Production host: Escherichia coli (E. coli)
References: UniProt: Q96PU5, HECT-type E3 ubiquitin transferase, RING-type E3 ubiquitin transferase (cysteine targeting)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Nedd4-2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 130 mM KCl, 20 mM NaCl, 1 mM EDTA, 1 mM TCEP, 3mM CHAPSO
SpecimenConc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.9.8model fitting
9cryoSPARC4.4.1initial Euler assignment
10cryoSPARC4.4.1final Euler assignment
11cryoSPARC4.4.1.classification
12cryoSPARC4.4.1.3D reconstruction
13PHENIX1.19.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225759 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12ONI

2oni

12ONI1PDBexperimental model
22NSQ

2nsq

12NSQ2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025464
ELECTRON MICROSCOPYf_angle_d0.4137391
ELECTRON MICROSCOPYf_dihedral_angle_d8.7392077
ELECTRON MICROSCOPYf_chiral_restr0.038764
ELECTRON MICROSCOPYf_plane_restr0.003966

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