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- PDB-7k48: Structure of NavAb/Nav1.7-VS2A chimera trapped in the resting sta... -

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Basic information

Entry
Database: PDB / ID: 7k48
TitleStructure of NavAb/Nav1.7-VS2A chimera trapped in the resting state by tarantula toxin m3-Huwentoxin-IV
Components
  • Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
  • Mu-theraphotoxin-Hs2a
KeywordsMEMBRANE PROTEIN / Ion channel / Voltage-gated sodium channel / Gating-modifier toxin
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / host cell presynaptic membrane / membrane depolarization during action potential / cardiac muscle cell action potential involved in contraction / voltage-gated sodium channel complex / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / ion channel inhibitor activity / detection of maltose stimulus / sodium ion transport ...detection of mechanical stimulus involved in sensory perception / host cell presynaptic membrane / membrane depolarization during action potential / cardiac muscle cell action potential involved in contraction / voltage-gated sodium channel complex / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / ion channel inhibitor activity / detection of maltose stimulus / sodium ion transport / maltose transport complex / behavioral response to pain / Phase 0 - rapid depolarisation / maltose binding / maltose transport / maltodextrin transmembrane transport / detection of temperature stimulus involved in sensory perception of pain / carbohydrate transport / carbohydrate transmembrane transporter activity / sodium channel regulator activity / sodium ion transmembrane transport / neuronal action potential / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / sensory perception of pain / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / post-embryonic development / response to toxic substance / Sensory perception of sweet, bitter, and umami (glutamate) taste / circadian rhythm / outer membrane-bounded periplasmic space / toxin activity / periplasmic space / inflammatory response / axon / DNA damage response / extracellular region / membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit ...Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Voltage-dependent channel domain superfamily / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Ion transport protein / Maltose/maltodextrin-binding periplasmic protein / Huwentoxin-IV / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Arcobacter butzleri (bacteria)
Homo sapiens (human)
Haplopelma schmidti (Chinese earth tiger)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWisedchaisri, G. / Tonggu, L. / Gamal El-Din, T.M. / McCord, E. / Zheng, N. / Catterall, W.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS015751 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35 NS111573 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL112808 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural Basis for High-Affinity Trapping of the Na1.7 Channel in Its Resting State by Tarantula Toxin.
Authors: Goragot Wisedchaisri / Lige Tonggu / Tamer M Gamal El-Din / Eedann McCord / Ning Zheng / William A Catterall /
Abstract: Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting ...Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaAb with voltage-shifting mutations and the toxin-binding site of human Na1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.
History
DepositionSep 15, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 20, 2021Group: 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

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Assembly

Deposited unit
A: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
B: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
C: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
D: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
E: Mu-theraphotoxin-Hs2a
F: Mu-theraphotoxin-Hs2a
G: Mu-theraphotoxin-Hs2a
H: Mu-theraphotoxin-Hs2a


Theoretical massNumber of molelcules
Total (without water)290,0758
Polymers290,0758
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18260 Å2
ΔGint-171 kcal/mol
Surface area55540 Å2

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Components

#1: Protein
Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Neuroendocrine sodium channel ...MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / Sodium channel protein type IX subunit alpha / Voltage-gated sodium channel subunit alpha Nav1.7


Mass: 68519.062 Da / Num. of mol.: 4 / Mutation: R398A,L506A,M513V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Arcobacter butzleri (strain RM4018) (bacteria), (gene. exp.) Homo sapiens (human)
Strain: K12, RM4018 / Gene: malE, b4034, JW3994, Abu_1752, SCN9A, NENA / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P0AEX9, UniProt: A8EVM5, UniProt: Q15858
#2: Protein/peptide
Mu-theraphotoxin-Hs2a / Mu-TRTX-Hs2a / Huwentoxin-4 / Huwentoxin-IV / HwTx-IV / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb ...Mu-TRTX-Hs2a / Huwentoxin-4 / Huwentoxin-IV / HwTx-IV / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb / HWTX-IVb / Huwentoxin-IVc / HWTX-IVc


Mass: 3999.777 Da / Num. of mol.: 4 / Mutation: E1G, E4G, Y33W / Source method: obtained synthetically / Source: (synth.) Haplopelma schmidti (Chinese earth tiger) / References: UniProt: P83303

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IVCOMPLEXall0MULTIPLE SOURCES
2NavAb/Nav1.7-VS2A chimeraCOMPLEX#11MULTIPLE SOURCES
3m3-Huwentoxin-IVCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Escherichia coli (E. coli)83333K12
22Arcobacter butzleri (bacteria)367737RM4018
32Human (human)9606
43Haplopelma schmidti (Chinese earth tiger)29017
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HClTrisNH2C(CH2OH)3HCl1
2100 mMsodium chlorideNaClSodium chloride1
30.006 %glycol-diosgenin (GDN)C56H92O251
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: m3-HwTx-IV was added in excess to the chimera at 8:1 stoichiometric molar ratio of toxin to channel.
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blot for 2.5-4.0 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 8.6 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 7168
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE
Image scansWidth: 3710 / Height: 3582 / Movie frames/image: 42 / Used frames/image: 2-42

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Processing

EM software
IDNameVersionCategory
2Leginon3.4image acquisition
4GctfCTF correction
7UCSF Chimera1.11.2model fitting
9RELION3initial Euler assignment
10cisTEM1.0.0-betafinal Euler assignment
11RELION3classification
12cisTEM1.0.0-beta3D reconstruction
13PHENIX1.14-3260model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1470000
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 501310 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6N4I

6n4i


Pdb chain-ID: A

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