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- PDB-6vxz: SthK P300A cyclic nucleotide-gated potassium channel in the close... -

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Basic information

Entry
Database: PDB / ID: 6vxz
TitleSthK P300A cyclic nucleotide-gated potassium channel in the closed state, in complex with cAMP
ComponentsSthK
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


monoatomic ion transmembrane transport / protein-containing complex binding / membrane
Similarity search - Function
: / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Ion channel / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain ...: / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Ion channel / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Chem-PGW / Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsSchmidpeter, P.A.M. / Rheinberger, J. / Nimigean, C.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124451 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088352 United States
American Heart Association18POST33960309 United States
CitationJournal: Nat Commun / Year: 2020
Title: Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel.
Authors: Philipp A M Schmidpeter / Jan Rheinberger / Crina M Nimigean /
Abstract: SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the ...SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.
History
DepositionFeb 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: SthK
B: SthK
C: SthK
D: SthK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,66332
Polymers204,3704
Non-polymers19,29328
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43730 Å2
ΔGint-176 kcal/mol
Surface area75740 Å2

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Components

#1: Protein
SthK


Mass: 51092.539 Da / Num. of mol.: 4 / Mutation: P300A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)
Strain: ATCC 700085 / DSM 6578 / Z-1203 / Gene: Spith_0644 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41 / References: UniProt: G0GA88
#2: Chemical
ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H12N5O6P
#3: Chemical...
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
Has ligand of interestN

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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: SthK P300A bound to cAMP / Type: COMPLEX / Details: SthK P300A was reconstituted into nanodiscs. / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41(DE3)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMpotassium chlorideKCl1
33 mMcAMP1
43 mMFos-Choline-8, Fluorinated1
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 45620 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 0.001 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 71 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2494
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Spherical aberration corrector: Microscope was modified with a Cs corrector
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
10RELION3.0-betainitial Euler assignment
11RELION3.0-betafinal Euler assignment
13RELION3.0-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 657943
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61275 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementHighest resolution: 3.42 Å

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