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- PDB-8f0q: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acyl... -

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Basic information

Entry
Database: PDB / ID: 8f0q
TitleStructure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
ComponentsSodium 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 / membrane depolarization during action potential / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / sodium ion transport / behavioral response to pain / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain ...detection of mechanical stimulus involved in sensory perception / membrane depolarization during action potential / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / sodium ion transport / behavioral response to pain / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / neuronal action potential / sodium ion transmembrane transport / sensory perception of pain / post-embryonic development / response to toxic substance / Sensory perception of sweet, bitter, and umami (glutamate) taste / circadian rhythm / inflammatory response / axon / plasma membrane
Similarity search - Function
Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / 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
beta-D-mannopyranose / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Chem-X7R / CHOLESTEROL HEMISUCCINATE / Sodium channel protein PaFPC1 / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Periplaneta americana (American cockroach)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,60412
Polymers184,4821
Non-polymers6,12211
Water1,40578
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 , 1 types, 1 molecules A

#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
Source method: isolated from a genetically manipulated source
Details: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Periplaneta americana (American cockroach)
Gene: SCN9A, NENA / Cell line (production host): 293 suspension cells / Production host: Homo sapiens (human) / References: UniProt: D0E0C2, UniProt: Q15858

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Sugars , 3 types, 4 molecules

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


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3DManpb1-3DGlcpNAcb1-3DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2/a3-b1_b3-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(3+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{[(3+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#3: Sugar ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-mannopyranoseCOMMON NAMEGMML 1.0
b-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: 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

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Non-polymers , 4 types, 85 molecules

#5: Chemical ChemComp-X7R / 5-cyclopropyl-4-({1-[(1S)-1-(3,5-dichlorophenyl)ethyl]piperidin-4-yl}methoxy)-2-fluoro-N-(methanesulfonyl)benzamide


Mass: 543.478 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H29Cl2FN2O4S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#7: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 78 / Source method: isolated from a natural source / Formula: H2O

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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: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
Type: COMPLEX
Details: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Entity ID: #1 / 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
2200 mMsodium chlorideNaCl1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was reconstituted into lipid nanodiscs (MSP1E3D1 in 3POPC:1POPE:1POPG) and was monodisperse. The sample was crosslinked with 0.05% glutaraldehyde for 10 minutes at RT, then ...Details: The sample was reconstituted into lipid nanodiscs (MSP1E3D1 in 3POPC:1POPE:1POPG) and was monodisperse. The sample was crosslinked with 0.05% glutaraldehyde for 10 minutes at RT, then quenched with 1M Tris pH7.0.
Specimen supportDetails: Grids were incubated with a thiol reactive, self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol (SPT-0011P6, SensoPath Technologies, ...Details: Grids were incubated with a thiol reactive, self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol (SPT-0011P6, SensoPath Technologies, Inc., Bozeman, MT). Grids were incubated with this self-assembled monolayer (SAM) solution for 24 hours and afterwards rinsed with EtOH.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0./1
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 FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 3 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k 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: 2253983
Details: template-matching particle picking with a 30A low-pass filtered template
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 795792
Details: A score threshold was applied, so that only the best-scoring particle images would be included in the 3D reconstruction at each cycle.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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