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- EMDB-36397: Cryo-EM structure of SV2A dimer in complex with BoNT/A2 Hc and le... -

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Basic information

Entry
Database: EMDB / ID: EMD-36397
TitleCryo-EM structure of SV2A dimer in complex with BoNT/A2 Hc and levetiracetam
Map data
Sample
  • Complex: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
    • Protein or peptide: Botulinum neurotoxin
  • Protein or peptide: Synaptic vesicle glycoprotein 2A
  • Ligand: (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsSynaptic vesicle / epilepsy / TRANSPORT PROTEIN
Function / homology
Function and homology information


regulation of gamma-aminobutyric acid secretion / Toxicity of botulinum toxin type F (botF) / Toxicity of botulinum toxin type D (botD) / Toxicity of botulinum toxin type E (botE) / Toxicity of botulinum toxin type A (botA) / synaptic vesicle priming / presynaptic active zone / transmembrane transporter activity / protein transmembrane transporter activity / GABA-ergic synapse ...regulation of gamma-aminobutyric acid secretion / Toxicity of botulinum toxin type F (botF) / Toxicity of botulinum toxin type D (botD) / Toxicity of botulinum toxin type E (botE) / Toxicity of botulinum toxin type A (botA) / synaptic vesicle priming / presynaptic active zone / transmembrane transporter activity / protein transmembrane transporter activity / GABA-ergic synapse / neuromuscular junction / metalloendopeptidase activity / synaptic vesicle membrane / intracellular calcium ion homeostasis / cell-cell junction / synaptic vesicle / toxin activity / neuron projection / neuronal cell body / glutamatergic synapse / dendrite / protein kinase binding / endoplasmic reticulum / proteolysis / zinc ion binding / extracellular region / plasma membrane
Similarity search - Function
Synaptic vesicle protein SV2 / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Sugar transporter, conserved site / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor-binding C-terminal ...Synaptic vesicle protein SV2 / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Sugar transporter, conserved site / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridium neurotoxin, N-terminal receptor binding / Major facilitator superfamily / Major Facilitator Superfamily / Kunitz inhibitor STI-like superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Botulinum neurotoxin / Synaptic vesicle glycoprotein 2A
Similarity search - Component
Biological speciesHomo sapiens (human) / Clostridium botulinum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsYamagata A
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP22H02564 Japan
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for antiepileptic drugs and botulinum neurotoxin recognition of SV2A.
Authors: Atsushi Yamagata / Kaori Ito / Takehiro Suzuki / Naoshi Dohmae / Tohru Terada / Mikako Shirouzu /
Abstract: More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. ...More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. Synaptic vesicle glycoprotein 2a (SV2A), a putative membrane transporter in the synaptic vesicles (SVs), has been identified as a target of LEV and BRV. SV2A also serves as a receptor for botulinum neurotoxin (BoNT), which is the most toxic protein and has paradoxically emerged as a potent reagent for therapeutic and cosmetic applications. Nevertheless, no structural analysis on AEDs and BoNT recognition by full-length SV2A has been available. Here we describe the cryo-electron microscopy structures of the full-length SV2A in complex with the BoNT receptor-binding domain, BoNT/A2 H and either LEV or BRV. The large fourth luminal domain of SV2A binds to BoNT/A2 H through protein-protein and protein-glycan interactions. LEV and BRV occupy the putative substrate-binding site in an outward-open conformation. A propyl group in BRV creates additional contacts with SV2A, explaining its higher binding affinity than that of LEV, which was further supported by label-free spectral shift assay. Numerous LEV derivatives have been developed as AEDs and positron emission tomography (PET) tracers for neuroimaging. Our work provides a structural framework for AEDs and BoNT recognition of SV2A and a blueprint for the rational design of additional AEDs and PET tracers.
History
DepositionJun 2, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36397.png

Downloads & links

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Map

FileDownload / File: emd_36397.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-1.4020705 - 2.0093093
Average (Standard dev.)-0.00043343814 (±0.031098079)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36397_msk_1.map
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Half map: #2

Fileemd_36397_half_map_1.map
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Half map: #1

Fileemd_36397_half_map_2.map
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Sample components

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Entire : SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic d...

EntireName: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
Components
  • Complex: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
    • Protein or peptide: Botulinum neurotoxin
  • Protein or peptide: Synaptic vesicle glycoprotein 2A
  • Ligand: (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic d...

SupramoleculeName: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Synaptic vesicle glycoprotein 2A

MacromoleculeName: Synaptic vesicle glycoprotein 2A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.774008 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDYKDDDDKE EGFRDRAAFI RGAKDIAKEV KKHAAKKVVK GLDRVQDEYS RRSYSRFEEE DDDDDFPAPS DGYYRGEGTQ DEEEGGASS DATEGHDEDD EIYEGEYQGI PRAESGGKGE RMADGAPLAG VRGGLSDGEG PPGGRGEAQR RKEREELAQQ Y EAILRECG ...String:
MDYKDDDDKE EGFRDRAAFI RGAKDIAKEV KKHAAKKVVK GLDRVQDEYS RRSYSRFEEE DDDDDFPAPS DGYYRGEGTQ DEEEGGASS DATEGHDEDD EIYEGEYQGI PRAESGGKGE RMADGAPLAG VRGGLSDGEG PPGGRGEAQR RKEREELAQQ Y EAILRECG HGRFQWTLYF VLGLALMADG VEVFVVGFVL PSAEKDMCLS DSNKGMLGLI VYLGMMVGAF LWGGLADRLG RR QCLLISL SVNSVFAFFS SFVQGYGTFL FCRLLSGVGI GGSIPIVFSY FSEFLAQEKR GEHLSWLCMF WMIGGVYAAA MAW AIIPHY GWSFQMGSAY QFHSWRVFVL VCAFPSVFAI GALTTQPESP RFFLENGKHD EAWMVLKQVH DTNMRAKGHP ERVF SVTHI KTIHQEDELI EIQSDTGTWY QRWGVRALSL GGQVWGNFLS CFGPEYRRIT LMMMGVWFTM SFSYYGLTVW FPDMI RHLQ AVDYASRTKV FPGERVEHVT FNFTLENQIH RGGQYFNDKF IGLRLKSVSF EDSLFEECYF EDVTSSNTFF RNCTFI NTV FYNTDLFEYK FVNSRLINST FLHNKEGCPL DVTGTGEGAY MVYFVSFLGT LAVLPGNIVS ALLMDKIGRL RMLAGSS VM SCVSCFFLSF GNSESAMIAL LCLFGGVSIA SWNALDVLTV ELYPSDKRTT AFGFLNALCK LAAVLGISIF TSFVGITK A APILFASAAL ALGSSLALKL PETRGQVLQ

UniProtKB: Synaptic vesicle glycoprotein 2A

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Macromolecule #2: Botulinum neurotoxin

MacromoleculeName: Botulinum neurotoxin / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum (bacteria)
Molecular weightTheoretical: 49.456191 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KNIVNTSILS IVYKKDDLID LSRYGAKINI GDRVYYDSID KNQIKLINLE SSTIEVILKN AIVYNSMYEN FSTSFWIKIP KYFSKINLN NEYTIINCIE NNSGWKVSLN YGEIIWTLQD NKQNIQRVVF KYSQMVNISD YINRWIFVTI TNNRLTKSKI Y INGRLIDQ ...String:
KNIVNTSILS IVYKKDDLID LSRYGAKINI GDRVYYDSID KNQIKLINLE SSTIEVILKN AIVYNSMYEN FSTSFWIKIP KYFSKINLN NEYTIINCIE NNSGWKVSLN YGEIIWTLQD NKQNIQRVVF KYSQMVNISD YINRWIFVTI TNNRLTKSKI Y INGRLIDQ KPISNLGNIH ASNKIMFKLD GCRDPRRYIM IKYFNLFDKE LNEKEIKDLY DSQSNSGILK DFWGNYLQYD KP YYMLNLF DPNKYVDVNN IGIRGYMYLK GPRGSVVTTN IYLNSTLYEG TKFIIKKYAS GNEDNIVRNN DRVYINVVVK NKE YRLATN ASQAGVEKIL SALEIPDVGN LSQVVVMKSK DDQGIRNKCK MNLQDNNGND IGFIGFHLYD NIAKLVASNW YNRQ VGKAS RTFGCSWEFI PVDDGWGESS L

UniProtKB: Botulinum neurotoxin

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Macromolecule #4: (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide

MacromoleculeName: (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide / type: ligand / ID: 4 / Number of copies: 1 / Formula: UKX
Molecular weightTheoretical: 170.209 Da

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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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

BufferpH: 7.5
Details: 20 mM Hepes (pH7.5), 150 mM NaCl, 0.001% LMNG, 0.0002% cholesterol hemisuccinate
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6518 / Average electron dose: 50.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6409122
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 425512
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 555697 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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