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- PDB-9z5q: HECT domain of NEDD4-2 complex with a targeted nanobody, nb.C11 -

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Basic information

Entry
Database: PDB / ID: 9z5q
TitleHECT domain of NEDD4-2 complex with a targeted nanobody, nb.C11
Components
  • E3 ubiquitin-protein ligase NEDD4-like
  • Nanobody C11 (nb.C11)
KeywordsLIGASE / Ubiquitin / targeted protein degradation / E3 ligase / Nanobody
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 transmembrane transport / negative regulation of potassium ion export across plasma membrane / negative regulation of protein localization to cell surface / positive regulation of dendrite extension / regulation of membrane repolarization / 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 transmembrane transport / negative regulation of potassium ion export across plasma membrane / negative regulation of protein localization to cell surface / positive regulation of dendrite extension / regulation of membrane repolarization / 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 / regulation of synapse organization / neuromuscular junction development / 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 / monoatomic ion transmembrane transport / ubiquitin-dependent protein catabolic process / 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 / WW domain / C2 domain profile. / 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 speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsAfriyie, E. / Clarke, O.B.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)RO1-HL121253 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)RO1-HL142111 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01-NS126850 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01-HL164319 United States
American Heart Association20PRE35210815 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)F31 DK118866 United States
American Heart AssociationPOST1019343 United States
CitationJournal: Nat Commun / Year: 2025
Title: Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.
Authors: Arden Darko-Boateng / Emmanuel Afriyie / Travis J Morgenstern / Sri Karthika Shanmugam / Xinle Zou / Yianni D Laloudakis / Papiya Choudhury / Meera Desai / Robert S Kass / Francesca Vallese ...Authors: Arden Darko-Boateng / Emmanuel Afriyie / Travis J Morgenstern / Sri Karthika Shanmugam / Xinle Zou / Yianni D Laloudakis / Papiya Choudhury / Meera Desai / Robert S Kass / Francesca Vallese / Oliver B Clarke / Henry M Colecraft /
Abstract: Targeted protein degradation/downregulation (TPD/TPDR) is a disruptive paradigm for developing therapeutics. <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit ion channels has not been demonstrated. NEDD4-2 E3 ligase regulates myriad ion channels, but its utility for TPD/TPDR is uncertain due to complex regulatory mechanisms. Here, we identify a nanobody that binds NEDD4-2 HECT domain without disrupting catalysis sites as revealed by cryo-electron microscopy and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels (Ca2.2; KCNQ1; and epithelial Na channel, ENaC, with a Liddle syndrome mutation) using divalent nanobodies (DiVas) strongly suppresses their surface density and function. Global proteomics indicates DiVa recruitment of endogenous NEDD4-2 to KCNQ1-YFP yields dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of NEDD4-2 recruitment for TPD/TPDR, validate ion channels as susceptible to this modality, and introduce a general method to generate ion channel inhibitors.
History
DepositionNov 12, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jan 28, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.1Jan 28, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nanobody C11 (nb.C11)
B: E3 ubiquitin-protein ligase NEDD4-like


Theoretical massNumber of molelcules
Total (without water)70,4162
Polymers70,4162
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: Antibody Nanobody C11 (nb.C11)


Mass: 17817.080 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pET24 / Details (production host): NYCOMPS expression plasmid / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS
#2: Protein E3 ubiquitin-protein ligase NEDD4-like / HECT-type E3 ubiquitin transferase NED4L / NEDD4.2 / Nedd4-2


Mass: 52598.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: HECT domain of NEDD4-2 / Source: (gene. exp.) Homo sapiens (human) / Gene: NEDD4L, KIAA0439, NEDL3 / Plasmid: pET24 / Details (production host): NYCOMPS expression plasmid / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS
References: UniProt: Q96PU5, HECT-type E3 ubiquitin transferase, RING-type E3 ubiquitin transferase (cysteine targeting)
Has protein modificationY

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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: HECT domain of NEDD4-2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.07 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET24
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and 1 mM EDTA, 0.043% CHAPS
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2150 mMSodium ChlorideNaCl1
31 mMTris (2-carboxyethyl)phosphine1
41 mMEthylenediaminetetraacetic acid1
50.043 %3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate1
SpecimenConc.: 11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse and of high purifty
Specimen supportDetails: The grid was only glow discharged / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Vitrification carried out at Argon atmosphere

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS / Details: Preliminary grid screening was performed manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.092 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7790
Details: Images were collected in movie-mode at 100 frames per second
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.26aparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXversion 1.9.dev202409060131 (2024-09-06)model fitting
8Coot0.9.6.ELmodel fitting
10PHENIXv1.21-5207model refinement
14cryoSPARCv4.43D reconstruction
CTF correctionDetails: We generated ab initio reconstruction model in CryoSPARC and use it in our pipeline
Type: NONE
Particle selectionNum. of particles selected: 1631714
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90979 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: The initial local fitting was done using UCSF Chimera and proceed to use real space fitting to complete the whole assembly in Coot
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue rangeSource nameTypeDetails
14BE8

4be8

B4BE8B596-9751596-975PDBexperimental model
22MPT

2mpt

B2MPTB541-5952541-595PDBexperimental model
3OtherotherColabFold
RefinementHighest resolution: 3.06 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034549
ELECTRON MICROSCOPYf_angle_d0.5036158
ELECTRON MICROSCOPYf_dihedral_angle_d6.535619
ELECTRON MICROSCOPYf_chiral_restr0.038634
ELECTRON MICROSCOPYf_plane_restr0.003800

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