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- EMDB-19066: TREK2 in OGNG/CHS detergent micelle with biparatopic inhibitory n... -

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Basic information

Entry
Database: EMDB / ID: EMD-19066
TitleTREK2 in OGNG/CHS detergent micelle with biparatopic inhibitory nanobody Nb6158
Map dataTREK2 with linked inhibitory nanobody in OGNG CHS detergent micelle at pH 7.5
Sample
  • Complex: Complex of TREK2 and inhibitory nanobody Nb6158
    • Protein or peptide: TREK2
    • Protein or peptide: Nb6158
KeywordsIon channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


TWIK related potassium channel (TREK) / Phase 4 - resting membrane potential / stabilization of membrane potential / potassium ion leak channel activity / outward rectifier potassium channel activity / monoatomic ion channel complex / potassium channel activity / potassium ion transmembrane transport / memory / signal transduction / plasma membrane
Similarity search - Function
Two pore domain potassium channel, TREK / Two pore domain potassium channel / Potassium channel domain / Ion channel
Similarity search - Domain/homology
Potassium channel subfamily K member 10
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.37 Å
AuthorsSmith KHM / Tucker SJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation.
Authors: Karin E J Rödström / Alexander Cloake / Janina Sörmann / Agnese Baronina / Kathryn H M Smith / Ashley C W Pike / Jackie Ang / Peter Proks / Marcus Schewe / Ingelise Holland-Kaye / Simon R ...Authors: Karin E J Rödström / Alexander Cloake / Janina Sörmann / Agnese Baronina / Kathryn H M Smith / Ashley C W Pike / Jackie Ang / Peter Proks / Marcus Schewe / Ingelise Holland-Kaye / Simon R Bushell / Jenna Elliott / Els Pardon / Thomas Baukrowitz / Raymond J Owens / Simon Newstead / Jan Steyaert / Elisabeth P Carpenter / Stephen J Tucker /
Abstract: Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic ...Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
History
DepositionDec 7, 2023-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19066.png

Downloads & links

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Map

FileDownload / File: emd_19066.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTREK2 with linked inhibitory nanobody in OGNG CHS detergent micelle at pH 7.5
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.261
Minimum - Maximum-0.40462807 - 0.53327763
Average (Standard dev.)0.0026648482 (±0.035020113)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19066_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19066_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19066_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 : Complex of TREK2 and inhibitory nanobody Nb6158

EntireName: Complex of TREK2 and inhibitory nanobody Nb6158
Components
  • Complex: Complex of TREK2 and inhibitory nanobody Nb6158
    • Protein or peptide: TREK2
    • Protein or peptide: Nb6158

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Supramolecule #1: Complex of TREK2 and inhibitory nanobody Nb6158

SupramoleculeName: Complex of TREK2 and inhibitory nanobody Nb6158 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: TREK2

MacromoleculeName: TREK2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGLQTVMKWK TVVAIFVVVV VYLVTGGLVF RALEQPFESS QKNTIALEKA EFLRDHVCVS PQELETLIQH ALDADNAGVS PIGNSSNNSS HWDLGSAFFF AGTVITTIGY GNIAPSTEGG KIFCILYAIF GIPLFGFLLA GIGDQLGTIF GKSIARVEKV FRKKQVSQTK ...String:
MGLQTVMKWK TVVAIFVVVV VYLVTGGLVF RALEQPFESS QKNTIALEKA EFLRDHVCVS PQELETLIQH ALDADNAGVS PIGNSSNNSS HWDLGSAFFF AGTVITTIGY GNIAPSTEGG KIFCILYAIF GIPLFGFLLA GIGDQLGTIF GKSIARVEKV FRKKQVSQTK IRVISTILFI LAGCIVFVTI PAVIFKYIEG WTALESIYFV VVTLTTVGFG DFVAGGNAGI NYREWYKPLV WFWILVGLAY FAAVLSMIGD WLRVLSKKTK EEVGEAENLY FQSHHHHHHH HHHDYKDDDD K

UniProtKB: Potassium channel subfamily K member 10

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Macromolecule #2: Nb6158

MacromoleculeName: Nb6158 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLVESGGG LVQAGDSLRV SCAGSGFTFT SYGMGWFRQA PGKEREFVAS INWNSNTAYA DSVRGRFTIS RDNAESMMYL QMNSLKPEDT AVYYCAATRA YSKPRVDSRH YDYWGQGTQV TVSSGGGGSG GGGSGGGGSQ VQLVESGGGL VQAGGSLRLS CAASGRAGSG ...String:
QVQLVESGGG LVQAGDSLRV SCAGSGFTFT SYGMGWFRQA PGKEREFVAS INWNSNTAYA DSVRGRFTIS RDNAESMMYL QMNSLKPEDT AVYYCAATRA YSKPRVDSRH YDYWGQGTQV TVSSGGGGSG GGGSGGGGSQ VQLVESGGGL VQAGGSLRLS CAASGRAGSG YAMGWFRQAP GKEREIVGAI SWSGDNTYYA DSVRGRVTIS RDYAQNTVYL QMNSLKPEDT AVYYCAADGR GNLRRGTAGR YVEYWGQGTQ VTVSSHHHHH HEPEA

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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

Concentration5.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMKClPotassium chloride
0.12 % w/vC27H52O12Octyl glucose neopentyl glycol
0.012 % w/vC35H61NO7Cholesteryl hemisuccinate tris salt
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 301660
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8qz1

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0) / Number images used: 17788
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
FSC plot (resolution estimation)

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