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- EMDB-28776: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the hybr... -

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Basic information

Entry
Database: EMDB / ID: EMD-28776
TitleStructure of VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305
Map dataDensity modified map used for model refinements
Sample
  • Complex: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305
    • Protein or peptide: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: N-[6-(cyclopentylmethoxy)-1,3-benzothiazol-2-yl]-4-{[(1S,2S)-2-(dimethylamino)cyclohexyl]amino}-2-fluorobenzene-1-sulfonamide
  • Ligand: water
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / 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 ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / 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 / sensory perception of pain / post-embryonic development / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to toxic substance / 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 / 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. ...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
Sodium channel protein PaFPC1 / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsKschonsak M / Jao CC / Arthur CP / Rohou AL / Bergeron P / Ortwine D / McKerall SJ / Hackos DH / Deng L / Chen J ...Kschonsak M / Jao CC / Arthur CP / Rohou AL / Bergeron P / Ortwine D / McKerall SJ / Hackos DH / Deng L / Chen J / Sutherlin D / Dragovich PS / Volgraf M / Wright MR / Payandeh J / Ciferri C / Tellis JC
Funding support1 items
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, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28776.png

Downloads & links

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Map

FileDownload / File: emd_28776.map.gz / Format: CCP4 / Size: 8.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map used for model refinements
Voxel sizeX=Y=Z: 0.838 Å
Density
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-8.131942 - 21.511806
Average (Standard dev.)2.2775083e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin130156128
Dimensions141120134
Spacing134141120
CellA: 112.292 Å / B: 118.158 Å / C: 100.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Full map

Fileemd_28776_additional_1.map
AnnotationFull map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_28776_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_28776_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor ...

EntireName: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305
Components
  • Complex: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305
    • Protein or peptide: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: N-[6-(cyclopentylmethoxy)-1,3-benzothiazol-2-yl]-4-{[(1S,2S)-2-(dimethylamino)cyclohexyl]amino}-2-fluorobenzene-1-sulfonamide
  • Ligand: water

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Supramolecule #1: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor ...

SupramoleculeName: VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Details: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Sodium channel protein PaFPC1,Sodium channel protein type 9 subun...

MacromoleculeName: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
type: protein_or_peptide / ID: 1
Details: Chimeric construct of human Nav1.7 VSD4 and the NavPaS channel from American cockroach Periplaneta americana
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 184.481906 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWSHPQFEKG GGSGGGSGGS AWSHPQFEKG GSGGDYKDDD DKGGSGGDYK DDDDKMADNS PLIREERQRL FRPYTRAMLT APSAQPAKE NGKTEENKDN SRDKGRGANK DRDGSAHPDQ ALEQGSRLPA RMRNIFPAEL ASTPLEDFDP FYKNKKTFVV V TKAGDIFR ...String:
MWSHPQFEKG GGSGGGSGGS AWSHPQFEKG GSGGDYKDDD DKGGSGGDYK DDDDKMADNS PLIREERQRL FRPYTRAMLT APSAQPAKE NGKTEENKDN SRDKGRGANK DRDGSAHPDQ ALEQGSRLPA RMRNIFPAEL ASTPLEDFDP FYKNKKTFVV V TKAGDIFR FSGEKSLWML DPFTPIRRVA ISTMVQPIFS YFIMITILIH CIFMIMPATQ TTYILELVFL SIYTIEVVVK VL ARGFILH PFAYLRDPWN WLDFLVTLIG YITLVVDLGH LYALRAFRVL RSWRTVTIVP GWRTIVDALS LSITSLKDLV LLL LFSLSV FALIGLQLFM GNLKHKCVKH FPADGSWGNF TDERWFNYTS NSSHWYIPDD WIEYPLCGNS SGAGMCPPGY TCLQ GYGGN PNYGYTSFDT FGWAFLSVFR LVTLDYWEDL YQLALRSAGP WHILFFIIVV FYGTFCFLNF ILAVVVMSYT HMVKR ADEE KAAERELKKE KKAASVANNT ANGQEQTTIE MNGDEAVVID NNDQAARQQS DPETPAPSVT QRLTDFLCVW DCCVPW QKL QGAIGAVVLS PFFELFIAVI IVLNITFMAL DHHDMNIEFE RILRTGNYIF TSIYIVEAVL KIIALSPKFY FKDSWNV FD FIIVVFAILE LGLEGVQGLS VFRSFRLLRV FRLAKFWPTL NNFMSVMTKS YGAFVNVMYV MFLLLFIFAI IGMQLFGM N YIDNMERFPD GDLPRWNFTD FLHSFMIVFR ALCGEWIESM WDCMLVGDWS CIPFFVAVFF VGNLVILNLL IALLLNNYG SFCTSPTSDE EDSKDEDALA QIVRIFKRFK PNLNAVKLSP MKPDSEDIVE SQEIQGNNIA DAEDVLAGEF PPDCCCNAFY KCFPSRPAR DSSVQRMWSN IRRVCFLLAK NKYFQKFVTA VLVITSVLLA LEDIYLPQRP VLVNITLYVD YVLTAFFVIE M IIMLFAVG FKKYFTSKWY WLDFIVVVAY LLNFVLMCAG IEALQTLRLL RVFRLFRPLS KVNGMQVVTS TLVEAVPHIF NV ILVGIFF WLVFAIMGVQ LFAGKFYKCV DENSTVLSHE ITMDRNDCLH ENYTWENSPM NFDHVGNAYL SLLQVATFKG WLQ IMNDAI DSREVHKQPI RETNIYMYLY FIFFIVFGSF FILKLFVCIL IDIFRQQRRK AEGLSATDSR TQLIYRRAVM RTMS AKPVK RIPKPGNKIQ GCIFDLVTNQ AFDISIMVLI CLNMVTMMVE KEGQSQHMTE VLYWINVVFI ILFTGECVLK LISLR HYYF TVGWNIFDFV VVIISIVGMF LADLIETYFV SPTLFRVIRL ARIGRILRLV KGAKGIRLLL LALRKALRTL FNVSFL LFV IMFVYAVFGM EFFMHIRDAG AIDDVYNFKT FGQSIILLFQ LATSAGWDGV YFAIANEEDC RAPDHELGYP GNCGSRA LG IAYLVSYLII TCLVVINMYA AVILDYVLEV YEDSKEGLTD DDYDMFFEVW QQFDPEATQY IRYDQLSELL EALQPPLQ V QKPNKYKILS MNIPICKDDH IFYKDVLEAL VKDVFSRRGS PVEAGDVQAP NVDEAEYKPV SSTLQRQREE YCVRLIQNA WRKHKQQN

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #4: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 4 / Number of copies: 5 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM / Discrete optimized protein energy

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: N-[6-(cyclopentylmethoxy)-1,3-benzothiazol-2-yl]-4-{[(1S,2S)-2-(d...

MacromoleculeName: N-[6-(cyclopentylmethoxy)-1,3-benzothiazol-2-yl]-4-{[(1S,2S)-2-(dimethylamino)cyclohexyl]amino}-2-fluorobenzene-1-sulfonamide
type: ligand / ID: 6 / Number of copies: 1 / Formula: X7L
Molecular weightTheoretical: 546.72 Da
Chemical component information

ChemComp-X7L:
N-[6-(cyclopentylmethoxy)-1,3-benzothiazol-2-yl]-4-{[(1S,2S)-2-(dimethylamino)cyclohexyl]amino}-2-fluorobenzene-1-sulfonamide

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 111 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
200.0 mMNaClSodium chloridesodium chloride
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300
Details: Grids incubated with a thiol reactive, self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol (SPT-0011P6, SensoPath Technologies, Inc., ...Details: Grids 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2741402
Details: template-matching particle picking with a 30A low-pass filtered template
Initial angle assignmentType: OTHER / Software - Name: cisTEM (ver. 1.02)
Final 3D classificationNumber classes: 100 / Avg.num./class: 27414 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: OTHER / Software - Name: cisTEM (ver. 1.02) / Details: No data beyond 3.0 A were used in the refinements
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX (ver. 1.20)
Details: A score threshold was applied so that only the best scoring particles are included in the 3D reconstruction at each cycle.
Number images used: 1201168

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8f0p:
Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the hybrid inhibitor GNE-1305

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