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- PDB-8vqc: Structure of the voltage-gated sodium channel NavPas from America... -

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Basic information

Entry
Database: PDB / ID: 8vqc
TitleStructure of the voltage-gated sodium channel NavPas from American Cockroach Periplaneta Americana in complex with scorpion alpha-toxin LqhaIT
Components
  • Alpha-insect toxin LqhaIT
  • Sodium channel protein PaFPC1
KeywordsMEMBRANE PROTEIN / NavPas / scorpion toxin / ion channel / voltage-gated sodium channel
Function / homology
Function and homology information


membrane depolarization during action potential / voltage-gated sodium channel complex / sodium channel inhibitor activity / voltage-gated monoatomic cation channel activity / voltage-gated sodium channel activity / neuronal action potential / defense response / toxin activity / extracellular region
Similarity search - Function
Scorpion long chain toxin / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain profile. / Scorpion long chain toxin/defensin / Scorpion toxin-like domain / Knottins / Knottin, scorpion toxin-like / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage gated sodium channel, alpha subunit ...Scorpion long chain toxin / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain profile. / Scorpion long chain toxin/defensin / Scorpion toxin-like domain / Knottins / Knottin, scorpion toxin-like / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage gated sodium channel, alpha subunit / Knottin, scorpion toxin-like superfamily / Voltage-gated cation channel calcium and sodium / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Sodium channel protein PaFPC1 / Alpha-insect toxin LqhaIT
Similarity search - Component
Biological speciesPeriplaneta americana (American cockroach)
Leiurus quinquestriatus hebraeus (scorpion)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsPhulera, S. / Khoshouei, M. / Whicher, J. / Weihofen, W.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other privatena United States
CitationJournal: Structure / Year: 2024
Title: Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels.
Authors: Swastik Phulera / Callum J Dickson / Christopher J Schwalen / Maryam Khoshouei / Samantha J Cassell / Yishan Sun / Tara Condos / Jonathan Whicher / Wilhelm A Weihofen /
Abstract: Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on ...Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an "S4 down" conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.
History
DepositionJan 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin ...citation / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sodium channel protein PaFPC1
H: Alpha-insect toxin LqhaIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,6479
Polymers191,2722
Non-polymers3,3757
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Sodium channel protein PaFPC1 / PaFPC1 / NavPaS


Mass: 183875.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Periplaneta americana (American cockroach)
Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: D0E0C2
#2: Protein Alpha-insect toxin LqhaIT / Lqh-alpha-IT / Alpha-IT


Mass: 7396.455 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Leiurus quinquestriatus hebraeus (scorpion)
Production host: Escherichia coli (E. coli) / Strain (production host): SHuffle T7 Express / References: UniProt: P17728
#3: Polysaccharide beta-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-3)-[beta-D-mannopyranose-(1-6)-beta-D- ...beta-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-3)-[beta-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-6DManpb1-3[DManpb1-6DManpb1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2-2-2-2/a4-b1_b4-c1_c3-d1_c6-f1_d6-e1_f6-g1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][b-D-Manp]{[(6+1)][b-D-Manp]{}}[(6+1)][b-D-Manp]{[(6+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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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: NavPas-LqhaIT / Type: COMPLEX
Details: Voltage-gated sodium channel NavPas from American Cockroach Periplaneta Americana in complex with scorpion alpha-toxin LqhaIT
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Periplaneta americana (American cockroach)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Details: 25 mM Tris pH 7.5, 50 mM NaCl, 0.1% (w/v) digitonin
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris(HOCH2)3CNH21
250 mMSodium ChlorideNaCl1
30.1 % w/vDigitoninC56H92O291
SpecimenConc.: 6.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample post SEC
Specimen supportDetails: Electron microscopy grids (Quantifoil Au, 200-mesh copper R1.2/1.3) were glow-discharged for 90 s using PELCO easiGlow
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Sample was applied on the grid in Vitrobot Mark IV chamber (Thermo Fisher Scientific), blotted for 4 secs with a blot force of 25 and then plunged into ethane

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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 defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
4Gctf1.06CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.2model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARC4.0.1final Euler assignment
13cryoSPARC4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164000 / Details: using Cryosparc / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: maximum likelihood
Atomic model buildingPDB-ID: 5x0m

5x0m


Pdb chain-ID: a / Accession code: 5x0m / Chain residue range: 47-1521 / Pdb chain residue range: 47-1521 / Source name: PDB / Type: experimental model

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