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- PDB-8jlc: Cryo-EM structure of SV2A in complex with BoNT/A2 Hc and levetiracetam -

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Basic information

Entry
Database: PDB / ID: 8jlc
TitleCryo-EM structure of SV2A in complex with BoNT/A2 Hc and levetiracetam
Components
  • Botulinum neurotoxin
  • Synaptic 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 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 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: Botulinum neurotoxin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,4136
Polymers133,2302
Non-polymers1,1834
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Synaptic vesicle glycoprotein 2A


Mass: 83774.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SV2A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7L0J3
#2: Protein Botulinum neurotoxin


Mass: 49456.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: D2KCK3
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical ChemComp-UKX / (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide / levetiracetam


Mass: 170.209 Da / Num. of mol.: 1 / 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 in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
Type: COMPLEX / Entity ID: #2 / 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
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: 50.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6518

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6409122
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 646723 / Symmetry type: POINT
RefinementResolution: 2.88→156.04 Å / Cor.coef. Fo:Fc: 0.841 / SU B: 9.765 / SU ML: 0.181 / ESU R: 0.345
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.39682 --
obs0.39682 122504 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 190.147 Å2
Refinement stepCycle: 1 / Total: 8189
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.0128388
ELECTRON MICROSCOPYr_bond_other_d00.0167615
ELECTRON MICROSCOPYr_angle_refined_deg1.3511.63511350
ELECTRON MICROSCOPYr_angle_other_deg0.4431.55817685
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.17351009
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.942546
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.722101419
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.070.21244
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.029484
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021824
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_it17.67918.3754045
ELECTRON MICROSCOPYr_mcbond_other17.67718.3764045
ELECTRON MICROSCOPYr_mcangle_it26.62427.5725051
ELECTRON MICROSCOPYr_mcangle_other26.62127.5725052
ELECTRON MICROSCOPYr_scbond_it16.85120.4044343
ELECTRON MICROSCOPYr_scbond_other16.84920.4044344
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other26.93229.7746300
ELECTRON MICROSCOPYr_long_range_B_refined46.32734728
ELECTRON MICROSCOPYr_long_range_B_other46.32734729
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.88→2.955 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.173 9069 -
obs--100 %

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