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- PDB-6vy0: SthK P300A cyclic nucleotide-gated potassium channel in a putativ... -

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Basic information

Entry
Database: PDB / ID: 6vy0
TitleSthK P300A cyclic nucleotide-gated potassium channel in a putative active state, in complex with cAMP
ComponentsSthK
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


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
Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila DSM 6578 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.68 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


Theoretical massNumber of molelcules
Total (without water)204,3704
Polymers204,3704
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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 DSM 6578 (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

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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: Low-populated state of SthK P300A reconstituted into nanodiscs. Due to the low resolution, side chains could not be assigned and all amino acids are modeled as alanine. The register is ...Details: Low-populated state of SthK P300A reconstituted into nanodiscs. Due to the low resolution, side chains could not be assigned and all amino acids are modeled as alanine. The register is maintained from the high-resolution structure of the closed state.
Entity ID: all / 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 FIELDBright-field microscopy / 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: 6.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19918 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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