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- EMDB-73821: HECT domain of NEDD4-2 complex with a targeted nanobody, nb.C11 -

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Basic information

Entry
Database: EMDB / ID: EMD-73821
TitleHECT domain of NEDD4-2 complex with a targeted nanobody, nb.C11
Map dataSharpened Map B-factor: -103.6
Sample
  • Complex: HECT domain of NEDD4-2
    • Protein or peptide: Nanobody C11 (nb.C11)
    • Protein or peptide: E3 ubiquitin-protein ligase NEDD4-like
KeywordsUbiquitin / targeted protein degradation / E3 ligase / Nanobody / LIGASE
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 / 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 export across plasma membrane / negative regulation of potassium ion transmembrane transport / 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 / 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 / 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 / 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 / C2 domain / WW/rsp5/WWP domain signature. / 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) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsAfriyie E / Clarke OB
Funding support United States, 7 items
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, 2025-
Header (metadata) releaseDec 17, 2025-
Map releaseDec 17, 2025-
UpdateDec 17, 2025-
Current statusDec 17, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73821.png

Downloads & links

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Map

FileDownload / File: emd_73821.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map B-factor: -103.6
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 262.912 Å		1.03 Å/pix.
x 256 pix.
= 262.912 Å		1.03 Å/pix.
x 256 pix.
= 262.912 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.027 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.8357247 - 1.4121597
Average (Standard dev.)0.000009343639 (±0.017163388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 262.912 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_73821_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_73821_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HECT domain of NEDD4-2

EntireName: HECT domain of NEDD4-2
Components
  • Complex: HECT domain of NEDD4-2
    • Protein or peptide: Nanobody C11 (nb.C11)
    • Protein or peptide: E3 ubiquitin-protein ligase NEDD4-like

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Supramolecule #1: HECT domain of NEDD4-2

SupramoleculeName: HECT domain of NEDD4-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 70 KDa

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Macromolecule #1: Nanobody C11 (nb.C11)

MacromoleculeName: Nanobody C11 (nb.C11) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 17.81708 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKYLLPTAAA GLLLLAAQPA MAMDIGINSD PQVQLQESGG GLVQAGGSLR LSCAASGNIS DVWYMGWYRQ APGKEREFVA GIAYGTSTY YADSVKGRFT ISRDNAKNTV YLQMNSLKPE DTAVYYCAVI ITRFVSYPRQ HTYWGQGTQV TVSSPLEHHH H HH

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Macromolecule #2: E3 ubiquitin-protein ligase NEDD4-like

MacromoleculeName: E3 ubiquitin-protein ligase NEDD4-like / type: protein_or_peptide / ID: 2 / Details: HECT domain of NEDD4-2 / Number of copies: 1 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.598484 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SYSLNPNDLG PLPPGWEERI HLDGRTFYID HNSKITQWED PRLQNPAITG PAVPYSREFK QKYDYFRKKL KKPADIPNRF EMKLHRNNI FEESYRRIMS VKRPDVLKAR LWIEFESEKG LDYGGVAREW FFLLSKEMFN PYYGLFEYSA TDNYTLQINP N SGLCNEDH ...String:
SYSLNPNDLG PLPPGWEERI HLDGRTFYID HNSKITQWED PRLQNPAITG PAVPYSREFK QKYDYFRKKL KKPADIPNRF EMKLHRNNI FEESYRRIMS VKRPDVLKAR LWIEFESEKG LDYGGVAREW FFLLSKEMFN PYYGLFEYSA TDNYTLQINP N SGLCNEDH LSYFTFIGRV AGLAVFHGKL LDGFFIRPFY KMMLGKQITL NDMESVDSEY YNSLKWILEN DPTELDLMFC ID EENFGQT YQVDLKPNGS EIMVTNENKR EYIDLVIQWR FVNRVQKQMN AFLEGFTELL PIDLIKIFDE NELELLMCGL GDV DVNDWR QHSIYKNGYC PNHPVIQWFW KAVLLMDAEK RIRLLQFVTG TSRVPMNGFA ELYGSNGPQL FTIEQWGSPE KLPR AHTCF NRLDLPPYET FEDLREKLLM AVENAQGFEG VDMDYKDDDD K

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

Concentration11 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mM4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMSodium ChlorideNaCl
1.0 mMTris (2-carboxyethyl)phosphine
1.0 mMEthylenediaminetetraacetic acid
0.043 %3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate

Details: 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and 1 mM EDTA, 0.043% CHAPS
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 40 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 260.0 kPa / Details: The grid was only glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Vitrification carried out at Argon atmosphere.
DetailsThe sample was monodisperse and of high purifty

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsPreliminary grid screening was performed manually
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 7790 / Average electron dose: 1.092 e/Å2
Details: Images were collected in movie-mode at 100 frames per second
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1631714
CTF correctionSoftware - Name: cryoSPARC
Details: We generated ab initio reconstruction model in CryoSPARC and use it in our pipeline
Type: NONE
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4) / Number images used: 90979
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

4be8

chain_id: B, residue_range: 596-975, source_name: PDB, initial_model_type: experimental model

2mpt

chain_id: B, residue_range: 541-595, source_name: PDB, initial_model_type: experimental model
source_name: Other, initial_model_type: otherColabFold
DetailsThe initial local fitting was done using UCSF Chimera and proceed to use real space fitting to complete the whole assembly in Coot
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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