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- EMDB-36394: Cryo-EM structure of SV2A LD4 in complex with BoNT/A2 Hc in the S... -

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Basic information

Entry
Database: EMDB / ID: EMD-36394
TitleCryo-EM structure of SV2A LD4 in complex with BoNT/A2 Hc in the SV2A-levetiracetam-BoNT/A2 Hc complex
Map data
Sample
  • Complex: SV2A in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
    • Protein or peptide: Synaptic vesicle glycoprotein 2A
    • Protein or peptide: Botulinum neurotoxin
  • 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 / synaptic vesicle membrane / metalloendopeptidase activity / intracellular calcium ion homeostasis / cell-cell junction / synaptic vesicle / toxin activity / neuron projection / neuronal cell body / dendrite / glutamatergic synapse / 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 / Major facilitator superfamily / Major Facilitator Superfamily / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease ...Synaptic vesicle protein SV2 / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Sugar transporter, conserved site / Major facilitator superfamily / Major Facilitator Superfamily / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridium neurotoxin, N-terminal receptor binding / 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: 2.82 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-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36394.png

Downloads & links

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Map

FileDownload / File: emd_36394.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.215
Minimum - Maximum-1.4364487 - 2.080151
Average (Standard dev.)-0.0009497438 (±0.038328253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36394_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_36394_half_map_1.map
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Half map: #2

Fileemd_36394_half_map_2.map
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Sample components

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Entire : SV2A in complex with BoNT/A2 Hc domain and anti-epileptic drug, l...

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

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Supramolecule #1: SV2A in complex with BoNT/A2 Hc domain and anti-epileptic drug, l...

SupramoleculeName: SV2A in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#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: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.797238 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
YASRTKVFPG ERVEHVTFNF TLENQIHRGG QYFNDKFIGL RLKSVSFEDS LFEECYFEDV TSSNTFFRNC TFINTVFYNT DLFEYKFVN SRLINSTFLH NKEGCPLDV

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: 1 / 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: 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 / N-Acetylglucosamine

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6409122
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 660976 / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 646723
FSC plot (resolution estimation)

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