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- PDB-8fz7: TpeA bound closed MthK-A88F mutant in nanodisc -

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Basic information

Entry
Database: PDB / ID: 8fz7
TitleTpeA bound closed MthK-A88F mutant in nanodisc
ComponentsCalcium-gated potassium channel MthK
KeywordsMEMBRANE PROTEIN / MTHK / TRANSPORT PROTEIN / ION CHANNEL / Blocker TpeA
Function / homology
Function and homology information


monoatomic cation transmembrane transporter activity / potassium ion transport / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Regulator of K+ conductance, N-terminal / TrkA-N domain / RCK N-terminal domain profile. / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. / Potassium channel domain / Ion channel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
: / Chem-PGW / 1-(tripentyl-$l^{4}-azanyl)pentane / Calcium-gated potassium channel MthK
Similarity search - Component
Biological speciesMethanothermobacter thermautotrophicus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsAgarwal, S. / Nimigean, C.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM088352 United States
Citation
Journal: Nat Chem Biol / Year: 2024
Title: Calcium-gated potassium channel blockade via membrane-facing fenestrations.
Authors: Chen Fan / Emelie Flood / Nattakan Sukomon / Shubhangi Agarwal / Toby W Allen / Crina M Nimigean /
Abstract: Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because ...Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.
#1: Journal: Nature / Year: 2020
Title: Ball-and-chain inactivation in a calcium-gated potassium channel.
Authors: Chen Fan / Nattakan Sukomon / Emelie Flood / Jan Rheinberger / Toby W Allen / Crina M Nimigean /
Abstract: Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present. In neurons, inactivation of both sodium and potassium channels is ...Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.
History
DepositionJan 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 3, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calcium-gated potassium channel MthK
C: Calcium-gated potassium channel MthK
E: Calcium-gated potassium channel MthK
G: Calcium-gated potassium channel MthK
B: Calcium-gated potassium channel MthK
D: Calcium-gated potassium channel MthK
F: Calcium-gated potassium channel MthK
H: Calcium-gated potassium channel MthK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)302,83115
Polymers299,4588
Non-polymers3,3737
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Calcium-gated potassium channel MthK


Mass: 37432.258 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanothermobacter thermautotrophicus (archaea)
Gene: mthK, MTH_1520 / Production host: Escherichia coli (E. coli) / References: UniProt: O27564
#2: 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.: 4 / Source method: obtained synthetically / Formula: C40H77O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Chemical ChemComp-YQ1 / 1-(tripentyl-$l^{4}-azanyl)pentane


Mass: 298.570 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H44N
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
Has ligand of interestY

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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: TpeA bound MthK A88F mutant in 0 Ca2+ / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Methanothermobacter thermautotrophicus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
SpecimenConc.: 7 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: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Details: BLOTTING FOR 2 S (BLOT FORCE 0)

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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 defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 58.9 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
10RELION4initial Euler assignment
11RELION4final Euler assignment
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105404 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6U6D

6u6d


Accession code: 6U6D / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00617208
ELECTRON MICROSCOPYf_angle_d0.51423238
ELECTRON MICROSCOPYf_dihedral_angle_d8.0246512
ELECTRON MICROSCOPYf_chiral_restr0.0492692
ELECTRON MICROSCOPYf_plane_restr0.0073004

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