Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants

Abstract

Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cryo-EM of SV2A and SV2B.
Fig. 2: Conformation and architecture of SV2A and SV2B.
Fig. 3: Anticonvulsant binding site.
Fig. 4: Molecular determinants of PSL binding.
Fig. 5: Comparison of the anticonvulsant binding site and ligand selectivity.
Fig. 6: Proposed mechanisms of ASM.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. The coordinates and associated volumes for the cryo-EM reconstruction of the SV2A transmembrane region, SV2A LD–8783 complex and SV2B datasets have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the accession codes 8UO9, 8UOA, 8UO8, EMD-42431, EMD-42432 and EMD-42430, respectively. The half-maps and masks used for refinement for each dataset have also been deposited in the EMDB. Source data are provided with this paper.

References

  1. Chang, B. S. & Lowenstein, D. H. Epilepsy. N. Engl. J. Med. 349, 1257–1266 (2003).

    Article  PubMed  Google Scholar 

  2. World Health Organization. Epilepsy. https://www.who.int/news-room/fact-sheets/detail/epilepsy (2023).

  3. Banerjee, P. N., Filippi, D. & Hauser, W. A. The descriptive epidemiology of epilepsy—a review. Epilepsy Res. 85, 31–45 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Löscher, W. & Klein, P. The pharmacology and clinical efficacy of antiseizure medications: from bromide salts to cenobamate and beyond. CNS Drugs 35, 935–963 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Buckley, K. & Kelly, R. B. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J. Cell Biol. 100, 1284–1294 (1985).

    Article  CAS  PubMed  Google Scholar 

  6. Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Muglia, P. et al. Padsevonil randomized Phase IIa trial in treatment-resistant focal epilepsy: a translational approach. Brain Commun. 2, fcaa183 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Wood, M. et al. Pharmacological profile of the novel antiepileptic drug candidate padsevonil: interactions with synaptic vesicle 2 proteins and the GABAA receptor. J. Pharmacol. Exp. Ther. 372, 1–10 (2020).

    Article  CAS  PubMed  Google Scholar 

  9. Rademacher, M. et al. Efficacy and safety of adjunctive padsevonil in adults with drug‐resistant focal epilepsy: results from two double‐blind, randomized, placebo‐controlled trials. Epilepsia Open 7, 758 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Feany, M. B., Lee, S., Edwards, R. H. & Buckley, K. M. The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell 70, 861–867 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Bindra, P. S., Knowles, R. & Buckley, K. M. Conservation of the amino acid sequence of SV2, a transmembrane transporter in synaptic vesicles and endocrine cells. Gene 137, 299–302 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Bajjalieh, S. M., Peterson, K., Linial, M. & Scheller, R. H. Brain contains two forms of synaptic vesicle protein 2. Proc. Natl Acad. Sci. USA 90, 2150–2154 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schivell, A. E., Mochida, S., Kensel-Hammes, P., Custer, K. L. & Bajjalieh, S. M. SV2A and SV2C contain a unique synaptotagmin-binding site. Mol. Cell. Neurosci. 29, 56–64 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Schivell, A. E., Batchelor, R. H. & Bajjalieh, S. M. Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin. J. Biol. Chem. 271, 27770–27775 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Lazzell, D. R., Belizaire, R., Thakur, P., Sherry, D. M. & Janz, R. SV2B regulates synaptotagmin 1 by direct interaction. J. Biol. Chem. 279, 52124–52131 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Kaempf, N. et al. Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles. Proc. Natl Acad. Sci. USA 112, 7297–7302 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Harper, C. B. et al. An epilepsy-associated SV2A mutation disrupts synaptotagmin-1 expression and activity-dependent trafficking. J. Neurosci. 40, 4586–4595 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang, N. et al. Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1. J. Neurosci. 35, 2492–2507 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Voleti, R., Jaczynska, K. & Rizo, J. Ca2+-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes. eLife 9, e57154 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhou, Q. et al. Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis. Nature 525, 62–67 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Südhof, T. C. A molecular machine for neurotransmitter release: synaptotagmin and beyond. Nat. Med. 19, 1227–1231 (2013).

    Article  PubMed  Google Scholar 

  22. Madeo, M., Kovács, A. D. & Pearce, D. A. The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae. J. Biol. Chem. 289, 33066–33071 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ciruelas, K., Marcotulli, D. & Bajjalieh, S. M. Synaptic vesicle protein 2: a multi-faceted regulator of secretion. Semin. Cell Dev. Biol. 95, 130–141 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bajjalieh, S. M., Frantz, G. D., Weimann, J. M., McConnell, S. K. & Scheller, R. H. Differential expression of synaptic vesicle protein 2 (SV2) isoforms. J. Neurosci. 14, 5223–5235 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Janz, R., Goda, Y., Geppert, M., Missler, M. & Südhof, T. C. SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release. Neuron 24, 1003–1016 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Paulussen, I. et al. SV2B defines a subpopulation of synaptic vesicles. J. Mol. Cell Biol. https://doi.org/10.1093/jmcb/mjad054 (2023).

  27. Dardou, D. et al. Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system. Brain Res. 1367, 130–145 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Crèvecœur, J. et al. Expression of SV2 isoforms during rodent brain development. BMC Neurosci. 14, 87 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Crowder, K. M. et al. Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A). Proc. Natl Acad. Sci. USA 96, 15268–15273 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Calame, D. G., Herman, I. & Riviello, J. J. A de novo heterozygous rare variant in SV2A causes epilepsy and levetiracetam-induced drug-resistant status epilepticus. Epilepsy Behav. Rep. 15, 100425 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Serajee, F. J. & Huq, A. M. Homozygous mutation in synaptic vesicle glycoprotein 2A gene results in intractable epilepsy, involuntary movements, microcephaly, and developmental and growth retardation. Pediatr. Neurol. 52, 642–646.e1 (2015).

    Article  PubMed  Google Scholar 

  32. Wang, D. et al. Levetiracetam-induced a new seizure type in a girl with a novel SV2A gene mutation. Clin. Neurol. Neurosurg. 181, 64–66 (2019).

    Article  PubMed  Google Scholar 

  33. Liu, Z. et al. Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2. Nat. Commun. 14, 2338 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gustafsson, R., Zhang, S., Masuyer, G., Dong, M. & Stenmark, P. Crystal structure of botulinum neurotoxin A2 in complex with the human protein receptor SV2C reveals plasticity in receptor binding. Toxins 10, 153 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Ciruelas, K., Marcotulli, D., Sullivan, J. M. & Bajjalieh, S. M. Levetiracetam inhibits SV2A-synaptotagmin interaction at synapses that lack SV2B. Preprint at bioRxiv https://doi.org/10.1101/640185 (2019).

  36. Nowack, A., Yao, J., Custer, K. L. & Bajjalieh, S. M. SV2 regulates neurotransmitter release via multiple mechanisms. Am. J. Physiol. Cell Physiol. 299, C960–C967 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee, J. et al. Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis. PLoS ONE 10, e0116589 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Nabulsi, N. B. et al. Synthesis and preclinical evaluation of 11C-UCB-J as a PET tracer for imaging the synaptic vesicle glycoprotein 2A in the brain. J. Nucl. Med. 57, 777–784 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Dong, M. et al. SV2 is the protein receptor for botulinum neurotoxin A. Science 312, 592–596 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Yeh, F. L. et al. SV2 mediates entry of tetanus neurotoxin into central neurons. PLoS Pathog. 6, e1001207 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Dong, M. et al. Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons. Mol. Biol. Cell 19, 5226–5237 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yao, G. et al. N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat. Struct. Mol. Biol. 23, 656–662 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kwon, S. E. & Chapman, E. R. Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles. J. Biol. Chem. 287, 35658–35668 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Reigada, D. et al. Control of neurotransmitter release by an internal gel matrix in synaptic vesicles. Proc. Natl Acad. Sci. USA 100, 3485–3490 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Suo, Y. et al. Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2. Nat. Struct. Mol. Biol. 30, 1001–1011 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Parker, J. L., Kato, T., Kuteyi, G., Sitsel, O. & Newstead, S. Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-023-01039-y (2023).

  47. Pravda, L. et al. MOLEonline: a web-based tool for analyzing channels, tunnels and pores (2018 update). Nucleic Acids Res. 46, W368–W373 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Drew, D., North, R. A., Nagarathinam, K. & Tanabe, M. Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem. Rev. 121, 5289–5335 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang, L. et al. Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field. J. Am. Chem. Soc. 137, 2695–2703 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Abel, R., Wang, L., Harder, E. D., Berne, B. J. & Friesner, R. A. Advancing drug discovery through enhanced free energy calculations. Acc. Chem. Res. 50, 1625–1632 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Chen, W. et al. Enhancing hit discovery in virtual screening through absolute protein-ligand binding free-energy calculations. J. Chem. Inf. Model. 63, 3171–3185 (2023).

    Article  CAS  PubMed  Google Scholar 

  52. Shi, J. et al. Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding. Biochem. Soc. Trans. 39, 1341–1347 (2011).

    Article  CAS  PubMed  Google Scholar 

  53. Wolfes, A. C. & Dean, C. The diversity of synaptotagmin isoforms. Curr. Opin. Neurobiol. 63, 198–209 (2020).

    Article  CAS  PubMed  Google Scholar 

  54. Yang, X.-F. & Rothman, S. M. Levetiracetam has a time- and stimulation-dependent effect on synaptic transmission. Seizure 18, 615–619 (2009).

    Article  PubMed  Google Scholar 

  55. Yang, X. et al. Brivaracetam augments short-term depression and slows vesicle recycling. Epilepsia 56, 1899–1909 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Lynch, B. A. et al. Visualization of SV2A conformations in situ by the use of protein tomography. Biochem. Biophys. Res. Commun. 375, 491–495 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Budzinski, K. L. et al. Large structural change in isolated synaptic vesicles upon loading with neurotransmitter. Biophys. J. 97, 2577–2584 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Daniels, V., Wood, M., Leclercq, K., Kaminski, R. M. & Gillard, M. Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays. Br. J. Pharmacol. 169, 1091–1101 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wood, M. D., Sands, Z. A., Vandenplas, C. & Gillard, M. Further evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein. Epilepsia 59, e147–e151 (2018).

    Article  CAS  PubMed  Google Scholar 

  60. Wood, M. D. & Gillard, M. Evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein. Epilepsia 58, 255–262 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Botermans, W. et al. SDI-118, a novel procognitive SV2A modulator: first-in-human randomized controlled trial including PET/fMRI assessment of target engagement. Front. Pharm. 13, 1066447 (2023).

    Article  Google Scholar 

  62. Provins, L. & Quesnel, Y. 2-oxo-1-imidazolidinyl imidazothiadiazole derivatives. Publication number WO2012/143117, international application number PCT/EP2012/001658 (2012).

  63. Provins, L. & Chanteux, H. 2-oxo-1-imidazolidinyl imidazothiadiazole derivatives. Publication number WO2019/011767, international application number PCT/EP2018/068201 (2019).

  64. Quesnel, Y. 2-oxo-1-pyrrolidinyl imidazothiadiazole derivatives. Publication number WO2011/047860, international application number PCT/EP2010/006434 (2011).

  65. Provins, L. Lead optimization of CYP3A4-driven metabolism: subtle structural changes, huge impact [Oral communication]. In 32nd Symposium on Medicinal Chemistry (2021).

  66. Rana, M. S., Wang, X. & Banerjee, A. An improved strategy for fluorescent tagging of membrane proteins for overexpression and purification in mammalian cells. Biochemistry 57, 6741–6751 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  69. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  70. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 1–8 (2021).

    Article  Google Scholar 

  73. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1069–1078 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Bordoli, L. et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1–13 (2009).

    Article  CAS  PubMed  Google Scholar 

  78. Wang, R. Y.-R. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  79. Coleman, J. A., Green, E. M. & Gouaux, E. X-ray structures and mechanism of the human serotonin transporter. Nature 532, 334–339 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Schrödinger Release 2023-3: Maestro (Schrödinger, LLC, New York, 2023).

  81. Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234 (2013).

    Article  PubMed  Google Scholar 

  82. Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    Article  CAS  PubMed  Google Scholar 

  83. Young, T., Abel, R., Kim, B., Berne, B. J. & Friesner, R. A. Motifs for molecular recognition exploiting hydrophobic enclosure in protein-ligand binding. Proc. Natl Acad. Sci. USA 104, 808–813 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Desmond Molecular Dynamics System (D. E. Shaw Research, 2023).

  85. Maestro-Desmond Interoperability Tools (Schrödinger, LLC, 2023).

  86. Lu, C. et al. OPLS4: improving force field accuracy on challenging regimes of chemical space. J. Chem. Theory Comput. 17, 4291–4300 (2021).

    Article  CAS  PubMed  Google Scholar 

  87. Lenselink, E. B. et al. Predicting binding affinities for GPCR ligands using free-energy perturbation. ACS Omega 1, 293–304 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Schrödinger Release 2023-3: Glide (Schrödinger, LLC, 2023).

  89. Wang, L. et al. Modeling local structural rearrangements using FEP/REST: application to relative binding affinity predictions of CDK2 inhibitors. J. Chem. Theory Comput. 9, 1282–1293 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Yang at Rosalind Franklin University Midwest Proteome Center (North Chicago, Illinois, USA) for technical support with proteomics (grants no. NIH NCRR S10 and no. HRSA C 76 HF03610-01-00). Cryo-EM microscopy at the University of Pittsburgh was supported by National Institutes of Health grants no. S10 OD025009 and no. S10 OD019995. This work was supported by a Young Investigator Grant from the Brain and Behavior Research Foundation to J.A.C. (grant no. 30153).

Author information

Authors and Affiliations

Authors

Contributions

A.M. performed expression and purification, grid preparation, cryo-EM data processing and analysis, and model building of the SV2A–8783-Nb complex. M.F.M. performed expression and purification, grid preparation, cryo-EM data processing and analysis, and model building of SV2B. Radioligand assays were performed by A.M. and M.F.M. for SV2A and SV2B, respectively. E.L., C.A., M.Y. and P.S.H. contributed to the selection of compounds for SV2A and to map and pose interpretation. L.P. and A. Hall developed compounds and contributed chemical expertise. M.P. facilitated the collaboration by establishing legal agreements. M.L. and A. Hillisch confirmed the pose of the compounds by FEP experiments. M.G. and C.W. characterized the compounds in vivo/in vitro. M.F.M. prepared figures with input from A.M. and J.A.C. A.M., M.F.M. and J.A.C wrote the first paper draft. All authors contributed to paper editing and preparation.

Corresponding authors

Correspondence to Peter S. Horanyi or Jonathan A. Coleman.

Ethics declarations

Competing interests

E.L., C.A., M.Y., L.P., A. Hall, M.P., M.L., A. Hillisch, C.W., M.G. and P.S.H. are employees of UCB Pharma. The other authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Axel Brunger, Qiangjun Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Sara Osman and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Sequence alignment and conservation of human SV2s.

a. Alignment of the SV2 protein family, showing absolutely conserved residues in green, SV2A disease associated mutants in red, conserved residues involved directly in binding UCB-2500 or PSL in purple, and non-conserved residues involved directly in binding UCB-2500 or PSL in pink or blue. b. ConSurf analysis of SV2A, with most conserved residues in maroon and least conserved residues in turquoise. c. ConSurf analysis of SV2B, with most conserved residues in maroon and least conserved residues in turquoise.

Extended Data Fig. 2 Purification and preliminary cryo-EM analysis of SV2s.

a. Construct design for SV2A and SV2B. SV2AΔ64 was more monodisperse and expressed at significantly higher levels and thus was used for all protein biochemistry and structural studies. Purification tags consisting of 10xHis and TwinStrep tags fused to mVenus was used to purify both proteins. The tags were fused to the C-terminus of SV2A and the N-terminus of SV2B with a 3C protease site for removal during purification. b. Size exclusion chromatography of the SV2A-8783-UCB-2500 complex, with Trp fluorescence size exclusion of protein fractions for cryo-EM shown in the inset. c. Size exclusion chromatography of the SV2B-PSL complex for cryo-EM. d. SDS-PAGE analysis of SV2A-8783 and SV2B. #: Fully glycosylated SV2. *: Immaturely glycosylated SV2. †: Proteolysis product of SV2B. ‡: 8783-Nb. Gel is representative of >6 independent experiments. e. 2D classification of various SV2A complexes showing differences in transmembrane features. f. Mass spectrometry analysis of SV2B. g. 3D reconstruction of SV2A-UCB-J fit to the AlphaFold predicted structure (yellow). h. 3D reconstruction of SV2A-UCB-J-8783 fit to the AlphaFold predicted structure. SV2A is shown in yellow and 8783-Nb in pink. i. Thermostability of mVenus-tagged SV2B with (right) and without PSL (left), as assessed by fluorescence size exclusion chromatography.

Source data

Extended Data Fig. 3 SV2A-8783-UCB-2500 cryo-EM data and processing.

A representative micrograph is shown with a scale bar of 120 nm. Two datasets were collected, composed of 24,211 and 21,528 micrographs, respectively. After patch motion correction and CTF estimation, a blob picker was used to pick particles. Particles were classified in 2D and an ab-initio reconstruction was used to generate templates. The resulting templates were used to re-pick particles from each dataset, resulting in a total of ~10 million particles for each dataset, which were re-extracted and binned to a smaller size to facilitate data processing. Heterogenous refinement generated several ‘decoy’ volumes and one volume with SV2A features. Particles from each dataset were combined at this stage, analyzed by several rounds of heterogenous, non-uniform, and local refinement. The resulting particle stack was processed in Relion using Bayesian Polishing, then processed by further rounds of 2D classification and CTF refinement. Local refinement with masks focusing on either the transmembrane region (3.3 Å final resolution) or the luminal domain (3.8 Å final resolution) was used to produce the final maps, which were sharpened with DeepEMhancer to improve map interpretability.

Extended Data Fig. 4 Interpretation of SV2A-8783-UCB-2500 map.

a. Modelling of all 12 transmembrane helices, the luminal domain-8783 Nb complex, and the intracellular domain helices of SV2A. b. Local resolution of the SV2A transmembrane region map. c. FSC curve of the SV2A transmembrane region map. d. Euler angle distribution of the SV2A transmembrane reconstruction. e. Map to model correlation of the SV2A model with the transmembrane SV2A map. f. FSC curve of the SV2A luminal domain-8783 Nb complex. g. Map to model correlation of the SV2A luminal domain-8783 Nb complex.

Extended Data Fig. 5 SV2B-PSL cryo-EM data collection and processing.

A representative micrograph is shown with a scale bar of 100 nm. 2D class averages from the final particle stack show features of transmembrane helices. 45,266 micrographs were collected, and the template picker along with templates from a previous data set was used to pick particles after patch motion correction and CTF estimation. Approximately 10.1 million particles were extracted and binned to a smaller size for data processing. Initial 2D classification and ab-initio reconstructions produced a volume with SV2B features, which was used for several rounds of heterogenous refinement with other ‘decoy’ volumes to remove false particle picks in 3D. Approximately 1.1 million particles with SV2B features after heterogenous refinement were re-extracted at a full box size (336 pixels), classified in 2D, and refined further using non-uniform refinement. Relion Bayesian Polishing, classification in 2D and 3D, and local refinement with a mask focusing on the protein density produced a final set of ~62k particles that was reconstructed to 3.2 Å resolution map.

Extended Data Fig. 6 Interpretation of SV2B-PSL map.

a. Modelling of all 12 transmembrane helices, the luminal domain, and the intracellular domain helices of SV2B. b. FSC curve of the SV2B-PSL map. c. Local resolution of the SV2B-PSL map. d. Euler angle distribution of the SV2B-PSL reconstruction. e. Map to model correlation of the SV2B-PSL model to the SV2B-PSL map.

Extended Data Fig. 7 Interbundle salt bridges, botulinum neurotoxin E-luminal domain interaction analysis, and SLC22 family intracellular domain comparison.

a. Alignment of the SV2A luminal domain with the crystal structure of the chimeric SV2A-SV2C luminal domain complexed with botulinum toxin serotype E (BoNTE, electrostatic surface), specific to SV2A and SV2B (PDB 7UIB). Y535 and Y557 are essential for BoNTE binding. Residues in the SV2A luminal domain provide charge complementarity to the BoNTE surface. b. The SV2B luminal domain aligned with 7UIB, residues in the SV2B luminal domain provide charge complementarity to BoNTE. Y478 and Y500 are important for BoNTE binding. c. The SV2C luminal domain (PDB 5JMC) aligned to 7UIB. Residues analogous to SV2A Y535/SV2B Y478 and SV2A Y557/SV2B Y500 are T521 and E543 in SV2C, which disrupt binding to BoNTE. d. Comparison with luminal domain structures (green: 5JMC; light blue: 7UIA; hot pink: 6ES1) with the cryo-EM structure of SV2B (light pink). e. A salt bridge between E194 and R473 in SV2A is observed above the closed luminal gate (spheres). D471 is conserved amongst all 3 SV2s. Sidechain density is shown in light blue. f. A salt bridge between K146 and D413 in SV2B is observed above the closed luminal gate (spheres), with water-like density near the two sidechains. Sidechain and water density is shown in light blue. g. Alignment of the SV2A (green) and SV2B (light blue) intracellular domains with the intracellular domains of human organic cation transporter 1 (pink, PDB 8ET7) and rat organic anion transporter 1 (tan, PDB 8BW7). h. A salt bridge between E282 and R682 in SV2A is observed below the closed intracellular gate. Sidechain density is shown in light blue. i. A salt bridge between E225 and R623 in SV2B is observed beneath the closed intracellular gating residues (spheres). Sidechain density is shown in light blue. j. In the UCB-2500 (pink sticks) binding site, a salt bridge is formed between D179 and K694, between the closed luminal and intracellular gates (spheres). Sidechain density is shown in light blue. k. In the PSL binding site, a salt bridge is formed by D122 and K635 in between the closed luminal and intracellular gates (spheres), with water-like density. Sidechain and water density is shown in light blue.

Extended Data Fig. 8 Detailed binding site and ligand pose analysis.

a. LigPlot of the UCB-2500 binding site in SV2A. Hydrogen bonds are depicted as green dashed lines. b. LigPlot of the PSL binding site in SV2B. Hydrogen bonds are depicted as green dashed lines. c. Comparison of two poses of UCB-2500 fit in the cryo-EM density. d. Comparison of two poses of PSL fit in the cryo-EM density. e. Perturbation map used to perform the FEP calculations. Similarities between pairs of ligands are shown in green f. 3H-UCB-J saturation binding experiments to wild-type and D670A (red squares) and W300A (blue triangles) SV2A, the data are mean ± s.e.m. Mutation of W300 or D670 to alanine abolishes 3H-UCB-J binding. The Kd of UCB-J was measured for wild-type SV2A (black circles, Kd = 1.5 ± 0.1 nM). g. BRV competition for wild type SV2B (black curve, Ki = 85 ± 22 uM), G240C (red curve, Ki = 80 ± 23 uM), and C600G (light blue curve, Ki = 149 ± 69 uM). Data shown in panels f,g are shown as mean ± s.e.m., exact values, the number of repeated independent experiments are reported in Supplementary Table 4.

Source data

Extended Data Fig. 9 Extra densities observed in the SV2A-8783-UCB-2500 and SV2B-PSL cryo-EM maps.

a. Lipid-like and cholesteryl hemisuccinate densities (light blue) observed close to the intracellular membrane side of SV2A. b. Cholesteryl hemisuccinate density (light blue) in SV2B. c. Glyco-diosgenin and lipid-like densities (light blue) near the luminal side of SV2B. d. Glyco-diosgenin and lipid-like densities (light blue) near the intracellular side of SV2B. e. Unknown density (light pink) observed between several sidechains (sidechain density in light blue) in SV2B, near the intracellular membrane interface. f. Comparison of the central site PSL density (PSL: blue sticks, PSL density: pink) with the unknown density located in the membrane interface site. g. Unknown density (light blue) located between several polar residues (grey sticks) close to the luminal side opening of the central cavity of SV2B, above the central PSL (light blue sticks) binding site. h. Water-like density (light blue, water oxygens as red spheres) in the SV2B near D413. i. Water-like density near G262 in SV2B. j. Water-like density near R462 in SV2B. k. Water-like density near E215 in SV2B. l. Water-like density near N631 in SV2B.

Extended Data Fig. 10 Syt1 is predicted to bind to the N-terminal region of SV2A and binding of various ligands to SV2A and the SV2A-Syt1 complex.

a. The AlphaFold predicted SV2A structure (grey) was aligned to the experimental SV2A structure (dark blue) and cryoEM density (transparent grey). The C2B domain crystal structure (PDB: 4V11) of Syt1 (cyan) with bound SV2A peptide was aligned to the AlphaFold predicted structure. The AlphaFold predicted structure of Syt1 (tan) was also aligned to the C2B Syt1 crystal structure. b. The SV2B experimental structure (dark blue) was aligned to the AlphaFold predicted structure (grey) and the cryoEM density (transparent grey). c. Binding of UCB-J to SV2A (black curve, Kd = 3.7 ± 1.4 nM) or SV2A-Syt1 complex (red curve, Kd = 4.7 ± 1.8 nM). d. Binding of PSL to SV2A (black curve, Kd = 35 ± 3 nM) or SV2A-Syt1 complex (red curve, Kd = 30 ± 4 nM). e. Competition binding of LEV to SV2A (black curve, Ki = 4.4 ± .7 µM) or SV2A-Syt1 complex (red curve, Ki = 3.4 ± .7 µM). f. Competition binding of BRV to SV2A (black curve, Ki = 317 ± 55 nM) or SV2A-Syt1 complex (red curve, Ki = 353 ± 204 nM). Data shown in panels c-f are shown as mean ± s.e.m., exact values, the number of repeated independent experiments are reported in Supplementary Table 4.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, Tables 1–4 and discussion.

Reporting Summary

Supplementary Data 1

Source data for Supplementary Fig. 2.

Source data

Source Data Fig. 1

Source data for Fig. 1c.

Source Data Fig. 4

Source data for Fig. 4.

Source Data Fig. 5

Source data for Fig. 5.

Source Data Fig. 6

Unprocessed gels and western blots.

Source Data Fig. 6

Source data for Fig. 6.

Source Data Extended Data Fig. 2

Unprocessed gel.

Source Data Extended Data Fig./Table 8

Source data for Extended Data Fig. 8.

Source Data Extended Data Fig./Table 10

Source data for Extended Data Fig. 10.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mittal, A., Martin, M.F., Levin, E.J. et al. Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01335-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41594-024-01335-1

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing