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- PDB-8jli: Cryo-EM structure of SV2A dimer in complex levetiracetam -

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Basic information

Entry
Database: PDB / ID: 8jli
TitleCryo-EM structure of SV2A dimer in complex levetiracetam
ComponentsSynaptic vesicle glycoprotein 2A
KeywordsTRANSPORT PROTEIN / Synaptic vesicle / epilepsy
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 / GABA-ergic synapse / neuromuscular junction ...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 / GABA-ergic synapse / neuromuscular junction / synaptic vesicle membrane / intracellular calcium ion homeostasis / cell-cell junction / synaptic vesicle / neuron projection / neuronal cell body / glutamatergic synapse / dendrite / protein kinase binding / endoplasmic reticulum / 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 / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
: / Synaptic vesicle glycoprotein 2A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsYamagata, A.
Funding support Japan, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Synaptic vesicle glycoprotein 2A
B: Synaptic vesicle glycoprotein 2A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,8884
Polymers167,5482
Non-polymers3402
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Synaptic vesicle glycoprotein 2A


Mass: 83774.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SV2A, KIAA0736, PSEC0174 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7L0J3
#2: Chemical ChemComp-UKX / (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide / levetiracetam


Mass: 170.209 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H14N2O2 / Feature type: SUBJECT OF INVESTIGATION
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: SV2A dimer in complex with anti-epileptic drug, levetiracetam
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Clostridium botulinum (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM Hepes (pH7.5), 150 mM NaCl, 0.001% LMNG, 0.0002% cholesterol hemisuccinate
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10001

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7Cootmodel fitting
9REFMACmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6435573
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 424198 / Symmetry type: POINT
RefinementResolution: 3.38→3.38 Å / Cor.coef. Fo:Fc: 0.875 / SU B: 34.417 / SU ML: 0.53 / ESU R: 0.683
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.42122 --
obs0.42122 77597 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 180.064 Å2
Refinement stepCycle: 1 / Total: 9248
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0129492
ELECTRON MICROSCOPYr_bond_other_d00.0168656
ELECTRON MICROSCOPYr_angle_refined_deg1.2511.62812840
ELECTRON MICROSCOPYr_angle_other_deg0.4331.55420012
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.41851168
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.321556
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.999101528
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0580.21400
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0210830
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022194
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it15.73217.1684684
ELECTRON MICROSCOPYr_mcbond_other15.73217.1684684
ELECTRON MICROSCOPYr_mcangle_it26.19125.7395848
ELECTRON MICROSCOPYr_mcangle_other26.1925.7395849
ELECTRON MICROSCOPYr_scbond_it15.54319.8214808
ELECTRON MICROSCOPYr_scbond_other15.54219.8234809
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other26.59128.8746993
ELECTRON MICROSCOPYr_long_range_B_refined52.27739552
ELECTRON MICROSCOPYr_long_range_B_other52.27739551
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.108 5660 -
obs--100 %

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