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- PDB-8f0s: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the hybr... -

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Basic information

Entry
Database: PDB / ID: 8f0s
TitleStructure of VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-9296
Components
  • Beta-diguetoxin-Dc1a
  • Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
KeywordsMEMBRANE PROTEIN/INHIBITOR / Ion channel / small molecule / inhibitor / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / behavioral response to pain / voltage-gated calcium channel complex / Phase 0 - rapid depolarisation ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / behavioral response to pain / voltage-gated calcium channel complex / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / calcium ion import across plasma membrane / sodium ion transmembrane transport / sodium channel regulator activity / sensory perception of pain / post-embryonic development / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to toxic substance / circadian rhythm / toxin activity / inflammatory response / axon / extracellular region / plasma membrane
Similarity search - Function
Beta/Mu-diguetoxin-1 / Spider Toxins mu-diguetoxin-1 a, b and c / 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-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage gated sodium channel, alpha subunit ...Beta/Mu-diguetoxin-1 / Spider Toxins mu-diguetoxin-1 a, b and c / 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-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-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 / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Chem-X80 / CHOLESTEROL HEMISUCCINATE / Sodium channel protein PaFPC1 / Beta-diguetoxin-Dc1a / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Periplaneta americana (American cockroach)
Diguetia canities (spider)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKschonsak, M. / Jao, C.C. / Arthur, C.P. / Rohou, A.L. / Bergeron, P. / Ortwine, D. / McKerall, S.J. / Hackos, D.H. / Deng, L. / Chen, J. ...Kschonsak, M. / Jao, C.C. / Arthur, C.P. / Rohou, A.L. / Bergeron, P. / Ortwine, D. / McKerall, S.J. / Hackos, D.H. / Deng, L. / Chen, J. / Sutherlin, D. / Dragovich, P.S. / Volgraf, M. / Wright, M.R. / Payandeh, J. / Ciferri, C. / Tellis, J.C.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Elife / Year: 2023
Title: Cryo-EM reveals an unprecedented binding site for Na1.7 inhibitors enabling rational design of potent hybrid inhibitors.
Authors: Marc Kschonsak / Christine C Jao / Christopher P Arthur / Alexis L Rohou / Philippe Bergeron / Daniel F Ortwine / Steven J McKerrall / David H Hackos / Lunbin Deng / Jun Chen / Tianbo Li / ...Authors: Marc Kschonsak / Christine C Jao / Christopher P Arthur / Alexis L Rohou / Philippe Bergeron / Daniel F Ortwine / Steven J McKerrall / David H Hackos / Lunbin Deng / Jun Chen / Tianbo Li / Peter S Dragovich / Matthew Volgraf / Matthew R Wright / Jian Payandeh / Claudio Ciferri / John C Tellis /
Abstract: The voltage-gated sodium (Na) channel Na1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na channel-blocking ...The voltage-gated sodium (Na) channel Na1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na channel-blocking drugs are not selective among the nine Na channel subtypes, Na1.1-Na1.9. Moreover, the two currently known classes of Na1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of Na1.7 inhibitors, exemplified by the clinical development candidate , has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the Na1.7 channel, we pursued high-resolution ligand-bound Na1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that engages the Na1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in Na channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective Na channel modulators targeting VSD4.
History
DepositionNov 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
B: Beta-diguetoxin-Dc1a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,74311
Polymers192,7222
Non-polymers4,0219
Water0
1


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

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera / / PaFPC1 / NavPaS / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / ...PaFPC1 / NavPaS / 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: 184481.906 Da / Num. of mol.: 1
Fragment: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Periplaneta americana (American cockroach)
Gene: SCN9A, NENA / Production host: Homo sapiens (human) / References: UniProt: D0E0C2, UniProt: Q15858
#2: Protein Beta-diguetoxin-Dc1a / Beta-DGTX-Dc1a / Dc1a / Insecticidal toxin DTX9.2


Mass: 8240.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Diguetia canities (spider) / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49126

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Sugars , 3 types, 6 molecules

#3: Polysaccharide beta-D-mannopyranose-(1-3)-[beta-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-3)-2-acetamido-2- ...beta-D-mannopyranose-(1-3)-[beta-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3[DManpb1-6]DManpb1-3DGlcpNAcb1-3DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2-2/a3-b1_b3-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(3+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{[(3+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.: 1
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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / 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

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Non-polymers , 3 types, 3 molecules

#6: Chemical ChemComp-X80 / 5-chloro-4-(cyclopentylmethoxy)-N-(4-{[(1S,2S)-2-(dimethylamino)cyclohexyl]amino}-2-fluorobenzene-1-sulfonyl)-2-fluorobenzamide


Mass: 570.091 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H34ClF2N3O4S / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#8: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4

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Details

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: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-9296
Type: COMPLEX
Details: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Periplaneta americana (American cockroach)6978
Source (recombinant)Organism: Homo sapiens (human) / Cell: 293 suspension cells
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
20.05 %digitonindigitonin1
30.005 %Cholesteryl HemisuccinateCHS1
450 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodisperse. The sample was crosslinked with 0.05% glutaraldehyde for 10 minutes at RT, then quenched with 1M Tris pH7.0.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 10 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEM3.7.11image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.2model refinement
10cisTEM1.02initial Euler assignment
11cisTEM1.02final Euler assignment
12RELION3.1classification
13PHENIX1.23D reconstruction
CTF correctionDetails: microgaphs with CTFfit of 6.0 A or better were selected
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1774062
Details: template-matching particle picking with a 30A low-pass filtered template
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 826525 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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