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- EMDB-28777: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acyl... -

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Basic information

Entry
Database: EMDB / ID: EMD-28777
TitleStructure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
Map dataDensity modified map used for model refinements
Sample
  • Complex: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
    • Protein or peptide: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
  • Ligand: beta-D-mannopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 5-cyclopropyl-4-({1-[(1S)-1-(3,5-dichlorophenyl)ethyl]piperidin-4-yl}methoxy)-2-fluoro-N-(methanesulfonyl)benzamide
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
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
Sodium channel protein PaFPC1 / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 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_28777.png

Downloads & links

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Map

FileDownload / File: emd_28777.map.gz / Format: CCP4 / Size: 8.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map used for model refinements
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 133 pix.
= 111.454 Å		0.84 Å/pix.
x 141 pix.
= 118.158 Å		0.84 Å/pix.
x 120 pix.
= 100.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.838 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-8.534427 - 21.089323
Average (Standard dev.)2.4730908e-11 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin130156129
Dimensions141120133
Spacing133141120
CellA: 111.454 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: non-modified, full map

Fileemd_28777_additional_1.map
Annotationnon-modified, full map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map1

Fileemd_28777_half_map_1.map
Annotationhalf-map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map2

Fileemd_28777_half_map_2.map
Annotationhalf-map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acyl...

EntireName: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
Components
  • Complex: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
    • Protein or peptide: Sodium channel protein PaFPC1,Sodium channel protein type 9 subunit alpha chimera
  • Ligand: beta-D-mannopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 5-cyclopropyl-4-({1-[(1S)-1-(3,5-dichlorophenyl)ethyl]piperidin-4-yl}methoxy)-2-fluoro-N-(methanesulfonyl)benzamide
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water

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Supramolecule #1: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acyl...

SupramoleculeName: Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310
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: beta-D-mannopyranose

MacromoleculeName: beta-D-mannopyranose / type: ligand / ID: 3 / Number of copies: 1 / Formula: BMA
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BMA:
beta-D-mannopyranose

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: 5-cyclopropyl-4-({1-[(1S)-1-(3,5-dichlorophenyl)ethyl]piperidin-4...

MacromoleculeName: 5-cyclopropyl-4-({1-[(1S)-1-(3,5-dichlorophenyl)ethyl]piperidin-4-yl}methoxy)-2-fluoro-N-(methanesulfonyl)benzamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: X7R
Molecular weightTheoretical: 543.478 Da
Chemical component information

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

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Macromolecule #6: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 6 / 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

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Macromolecule #7: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 78 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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 chloride
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300
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, ...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.
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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2253983
Details: template-matching particle picking with a 30A low-pass filtered template
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.5 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 particle images would be included in the 3D reconstruction at each cycle.
Number images used: 795792
Initial angle assignmentType: OTHER / Software - Name: cisTEM (ver. 1.02)
Final angle assignmentType: OTHER / Software - Name: cisTEM (ver. 1.02) / Details: No data beyond 3.4 A were used in the refinements
Final 3D classificationNumber classes: 100 / Software - Name: RELION (ver. 3.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8f0q:
Structure of VSD4-NaV1.7-NaVPas channel chimera bound to the acylsulfonamide inhibitor GDC-0310

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