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- PDB-8vtb: SthK R120A R124A in the presence of PIP2 and cAMP -

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Basic information

Entry
Database: PDB / ID: 8vtb
TitleSthK R120A R124A in the presence of PIP2 and cAMP
ComponentsTranscriptional regulator, Crp/Fnr family
KeywordsTRANSPORT PROTEIN / cyclic-nucleotide binding / potassium channel / lipid modulation
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 / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsSchmidpeter, P.A.M. / Thon, O. / Nimigean, C.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124451 United States
CitationJournal: Nat Commun / Year: 2024
Title: PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK.
Authors: Oliver Thon / Zhihan Wang / Philipp A M Schmidpeter / Crina M Nimigean /
Abstract: The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the ...The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.
History
DepositionJan 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 2, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcriptional regulator, Crp/Fnr family
B: Transcriptional regulator, Crp/Fnr family
C: Transcriptional regulator, Crp/Fnr family
D: Transcriptional regulator, Crp/Fnr family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)222,56332
Polymers202,3564
Non-polymers20,20828
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transcriptional regulator, Crp/Fnr family


Mass: 50588.918 Da / Num. of mol.: 4 / Mutation: R120A R124A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spirochaeta thermophila (bacteria) / Gene: Spith_0644 / Production host: Escherichia coli BL21(DE3) (bacteria) / 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-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, 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: tetrameric SthK R120A R124A protein / Type: COMPLEX
Details: SthK R120A R124A in complex with cAMP reconstituted into MSP1E3 nanodiscs containing PIP2
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Spirochaeta thermophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41 (DE3)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
110 mMHepes1
2100 mMKCl1
33 mMcAMP1
43 mMf-Foscholine 81
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: nanodisc reconstitution was carried out in the presence of DOPC, POPG, and PIP2
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 30 s incubation 2 s blotting blot force -4

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 56.64 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7particle selection
2Leginonimage acquisition
4CTFFIND4.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1799891
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 431535 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6CJQ

6cjq


Accession code: 6CJQ / Source name: PDB / Type: experimental model

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