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- PDB-8qz3: Crystal structure of human two pore domain potassium ion channel ... -

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Basic information

Entry
Database: PDB / ID: 8qz3
TitleCrystal structure of human two pore domain potassium ion channel TREK-2 (K2P10.1) in complex with an activatory nanobody (Nb67)
Components
  • Nanobody 67
  • 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) / cellular response to arachidonate / mechanosensitive potassium channel activity / Phase 4 - resting membrane potential / potassium ion leak channel activity / outward rectifier potassium channel activity / monoatomic ion channel complex / potassium channel activity / potassium ion transmembrane transport / 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: 2.4 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
Revision 1.1Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Potassium channel subfamily K member 10
B: Potassium channel subfamily K member 10
C: Nanobody 67
D: Nanobody 67
E: Nanobody 67
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,46111
Polymers105,1475
Non-polymers3146
Water3,441191
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The third nanobody might be present due to crystal packing.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16810 Å2
ΔGint-142 kcal/mol
Surface area41280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)51.264, 102.1, 215.817
Angle α, β, γ (deg.)90, 90, 90
Int Tables number18
Space group name H-MP22121

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Components

#1: Protein Potassium channel subfamily K member 10 / Outward rectifying potassium channel protein TREK-2 / TREK-2 K(+) channel subunit


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, TREK2 / Plasmid: pFB-CT10HF-LIC / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P57789
#2: Antibody Nanobody 67


Mass: 14944.368 Da / Num. of mol.: 3
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.: 5 / Source method: obtained synthetically / Formula: K
#4: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 191 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: 2.69 Å3/Da / Density % sol: 54.31 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.1 M Tris pH 8.0, 65% v/v 2-methyl-2,4-pentanediol

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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: Feb 3, 2019
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 2.4→58.808 Å / Num. obs: 34388 / % possible obs: 75.6 % / Redundancy: 6.4 % / CC1/2: 0.989 / Rpim(I) all: 0.087 / Rrim(I) all: 0.219 / Net I/σ(I): 6.3
Reflection shellResolution: 2.4→2.492 Å / Redundancy: 6.3 % / Mean I/σ(I) obs: 1.6 / Num. unique obs: 1719 / CC1/2: 0.61 / Rpim(I) all: 0.536 / Rrim(I) all: 1.363 / % possible all: 35.9

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Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSdata reduction
Aimlessdata scaling
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.4→58.81 Å / Cor.coef. Fo:Fc: 0.88 / Cor.coef. Fo:Fc free: 0.877 / SU R Cruickshank DPI: 0.793 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.818 / SU Rfree Blow DPI: 0.344 / SU Rfree Cruickshank DPI: 0.348
RfactorNum. reflection% reflectionSelection details
Rfree0.2909 1746 -RANDOM
Rwork0.2775 ---
obs0.2782 34319 75.6 %-
Displacement parametersBiso mean: 44.35 Å2
Baniso -1Baniso -2Baniso -3
1-11.9984 Å20 Å20 Å2
2---3.892 Å20 Å2
3----8.1065 Å2
Refine analyzeLuzzati coordinate error obs: 0.46 Å
Refinement stepCycle: LAST / Resolution: 2.4→58.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6864 0 13 191 7068
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0057050HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.689592HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2284SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes1187HARMONIC5
X-RAY DIFFRACTIONt_it7050HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion925SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact5576SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.03
X-RAY DIFFRACTIONt_other_torsion14.55
LS refinement shellResolution: 2.4→2.44 Å
RfactorNum. reflection% reflection
Rfree0.335 30 -
Rwork0.3464 --
obs0.3458 687 33.64 %
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)
10.82030.0127-1.38470.9240.11953.77870.08540.00450.36880.0045-0.18060.13420.36880.13420.0952-0.0767-0.0231-0.0594-0.09290.0892-0.081110.39813.625747.6975
20.41750.03690.88071.0643-0.12614.4060.0862-0.0175-0.4063-0.0175-0.02790.4859-0.40630.4859-0.0583-0.0501-0.05590.0708-0.04710.0625-0.125714.321818.270747.6142
32.5311-0.8859-0.27894.5504-1.62184.2664-0.03530.16030.00480.16030.13680.08390.00480.0839-0.1014-0.1492-0.059-0.0147-0.3248-0.005-0.3360.879835.8145.9232
40.9293-0.30940.48984.6215-3.12059.27570.0055-0.08950.0078-0.0895-0.0990.31520.00780.31520.0934-0.0936-0.04610.0279-0.1876-0.0607-0.30435.933510.873984.6263
51.59490.44660.79426.36262.89873.8955-0.01240.0340.31490.0340.20440.30530.31490.3053-0.1920.09840.1623-0.0472-0.16440.0514-0.076719.4193-12.766710.0062
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|74 - A|333 A|401 - A|403 B|601 - B|602 }A74 - 333
2X-RAY DIFFRACTION1{ A|74 - A|333 A|401 - A|403 B|601 - B|602 }A401 - 403
3X-RAY DIFFRACTION1{ A|74 - A|333 A|401 - A|403 B|601 - B|602 }B601 - 602
4X-RAY DIFFRACTION2{ B|74 - B|339 }B74 - 339
5X-RAY DIFFRACTION3{ C|1 - C|127 }C1 - 127
6X-RAY DIFFRACTION4{ D|1 - D|127 }D1 - 127
7X-RAY DIFFRACTION5{ E|1 - E|126 }E1 - 126

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