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- PDB-8qz2: Crystal structure of human two pore domain potassium ion channel ... -

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Basic information

Entry
Database: PDB / ID: 8qz2
TitleCrystal structure of human two pore domain potassium ion channel TREK-2 (K2P10.1) in complex with an inhibitory nanobody (Nb61)
Components
  • Nanobody 61Single-domain antibody
  • Potassium channel subfamily K member 10
KeywordsMEMBRANE PROTEIN / Potassium ion channel / Nanobody / Structural Genomics / Structural Genomics Consortium / SGC
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.5 Å
AuthorsBaronina, A. / Pike, A.C.W. / Rodstrom, K.E.J. / Ang, J. / Bushell, S.R. / Chalk, R. / Mukhopadhyay, S.M.M. / Pardon, E. / Arrowsmith, C.H. / Edwards, A.M. ...Baronina, A. / Pike, A.C.W. / Rodstrom, K.E.J. / Ang, J. / Bushell, S.R. / Chalk, R. / Mukhopadhyay, S.M.M. / Pardon, E. / Arrowsmith, C.H. / Edwards, A.M. / Bountra, C. / Burgess-Brown, N.A. / Tucker, S.J. / Steyaert, J. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, France, Belgium, 8items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T002018/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S008608/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/W017741/1 United Kingdom
Grenoble Instruct-ERIC Center (ISBG) France
Research Foundation - Flanders (FWO) Belgium
Wellcome Trust106169/Z/14/Z United Kingdom
Wellcome TrustWT084655MA United Kingdom
Wellcome Trust102161/B/13/Z 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
DepositionOct 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium channel subfamily K member 10
B: Potassium channel subfamily K member 10
C: Nanobody 61
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,4757
Polymers75,3193
Non-polymers1564
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, One Nb61 per TREK-2 dimer. Crystal packing might prevent two Nb61 per dimer.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12480 Å2
ΔGint-100 kcal/mol
Surface area27170 Å2
Unit cell
Length a, b, c (Å)56.538, 111.698, 289.208
Angle α, β, γ (deg.)90, 90, 90
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Potassium channel subfamily K member 10


Mass: 30157.029 Da / Num. of mol.: 2 / Mutation: N149Q, N152Q, N153Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNK10 / Plasmid: pFB-CT10HF-LIC / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P57789
#2: Antibody Nanobody 61 / Single-domain antibody


Mass: 15004.447 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pMESy4 / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#3: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.04 Å3/Da / Density % sol: 59.5 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 4.5
Details: MAG7.9, 0.1 M Na acetate pH 4.5, 0.3 M KCl, 23% v/v polyethylene glycol 400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9686 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 16, 2017
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 3.499→57.842 Å / Num. obs: 11445 / % possible obs: 95 % / Redundancy: 5.6 % / CC1/2: 0.997 / Rpim(I) all: 0.223 / Rrim(I) all: 0.54 / Net I/σ(I): 3.7
Reflection shellResolution: 3.499→3.577 Å / Redundancy: 6.1 % / Mean I/σ(I) obs: 1.2 / Num. unique obs: 572 / CC1/2: 0.488 / Rpim(I) all: 0.799 / Rrim(I) all: 1.997 / % possible all: 79.7

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Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSdata reduction
Aimlessdata scaling
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.5→52.1 Å / Cor.coef. Fo:Fc: 0.798 / Cor.coef. Fo:Fc free: 0.792 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.675
RfactorNum. reflection% reflectionSelection details
Rfree0.3082 574 -RANDOM
Rwork0.2795 ---
obs0.2809 11398 95 %-
Displacement parametersBiso mean: 65.62 Å2
Baniso -1Baniso -2Baniso -3
1--1.5723 Å20 Å20 Å2
2--2.6622 Å20 Å2
3----1.0899 Å2
Refine analyzeLuzzati coordinate error obs: 0.59 Å
Refinement stepCycle: LAST / Resolution: 3.5→52.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4507 0 4 0 4511
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0054622HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.686316HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1419SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes769HARMONIC5
X-RAY DIFFRACTIONt_it4622HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion635SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact3586SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.02
X-RAY DIFFRACTIONt_other_torsion16.18
LS refinement shellResolution: 3.5→3.56 Å
RfactorNum. reflection% reflection
Rfree0.3396 25 -
Rwork0.3298 --
obs0.3303 408 78.33 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.42580.0761-0.42211.6621-0.52312.09270.11690.20190.44270.20190.0065-0.46640.4427-0.4664-0.12340.03210.0285-0.0230.1010.04980.1-4.22918.1096321.779
21.04860.0140.24011.1102-0.17963.28360.07150.28980.21830.28980.11170.27660.21830.2766-0.1832-0.11360.060.0263-0.02120.0918-0.08465.051917.471322.892
33.61031.51310.40244.9754-2.41168.66660.05480.1752-0.38340.17520.08780.2127-0.38340.2127-0.1426-0.31020.05090.0319-0.1639-0.0339-0.204811.124235.088296.566
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|74 - A|323 B|600 - B|603 }A74 - 323
2X-RAY DIFFRACTION1{ A|74 - A|323 B|600 - B|603 }B600 - 603
3X-RAY DIFFRACTION2{ B|75 - B|324 }B75 - 324
4X-RAY DIFFRACTION3{ C|2 - C|122 }C2 - 122

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