{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T13:17:11Z","timestamp":1792675031988,"version":"4.5.0"},"reference-count":44,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2021,8,2]],"date-time":"2021-08-02T00:00:00Z","timestamp":1627862400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004359","name":"Vetenskapsr\u00e5det","doi-asserted-by":"publisher","award":["2018-03406"],"award-info":[{"award-number":["2018-03406"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002794","name":"Cancerfonden","doi-asserted-by":"publisher","award":["20 1287 PjF"],"award-info":[{"award-number":["20 1287 PjF"]}],"id":[{"id":"10.13039\/501100002794","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT\/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT\/E and a significant rearrangement of loop 1228\u20131237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT\/E for complex gangliosides.<\/jats:p>","DOI":"10.3390\/ijms22158315","type":"journal-article","created":{"date-parts":[[2021,8,3]],"date-time":"2021-08-03T08:16:39Z","timestamp":1627978599000},"page":"8315","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9527-2310","authenticated-orcid":false,"given":"Geoffrey","family":"Masuyer","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"},{"name":"Centre for Therapeutic Innovation, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4986-8594","authenticated-orcid":false,"given":"Jonathan R.","family":"Davies","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4777-3417","authenticated-orcid":false,"given":"P\u00e5l","family":"Stenmark","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden"},{"name":"Department of Experimental Medical Science, Lund University, 22184 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1152\/physrev.2000.80.2.717","article-title":"Neurotoxins affecting neuroexocytosis","volume":"80","author":"Schiavo","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Fonfria, E., Maignel, J., Lezmi, S., Martin, V., Splevins, A., Shubber, S., Kalinichev, M., Foster, K., Picaut, P., and Krupp, J. (2018). The Expanding Therapeutic Utility of Botulinum Neurotoxins. Toxins, 10.","DOI":"10.3390\/toxins10050208"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/164_2020_351","article-title":"Novel Native and Engineered Botulinum Neurotoxins","volume":"263","author":"Steward","year":"2021","journal-title":"Handb. Exp. Pharmacol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Williamson, C.H., Sahl, J.W., Smith, T.J., Xie, G., Foley, B.T., Smith, L.A., Fern\u00e1ndez, R.A., Lindstr\u00f6m, M., Korkeala, H., and Keim, P. (2016). Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia. BMC Genom., 17.","DOI":"10.1186\/s12864-016-2502-z"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"14130","DOI":"10.1038\/ncomms14130","article-title":"Identification and characterization of a novel botulinum neurotoxin","volume":"8","author":"Zhang","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Peck, M.W., Smith, T.J., Anniballi, F., Austin, J.W., Bano, L., Bradshaw, M., Cuervo, P., Cheng, L.W., Derman, Y., and Dorner, B.G. (2017). Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature. Toxins, 9.","DOI":"10.3390\/toxins9010038"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/164_2019_342","article-title":"The Structure and Classification of Botulinum Toxins","volume":"263","author":"Dong","year":"2021","journal-title":"Handb. Exp. Pharm."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1146\/annurev-biochem-013118-111654","article-title":"Botulinum and Tetanus Neurotoxins","volume":"88","author":"Dong","year":"2019","journal-title":"Annu. Rev. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1126\/science.1161748","article-title":"Membrane fusion: Grappling with SNARE and SM proteins","volume":"323","author":"Rothman","year":"2009","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1146\/annurev.biochem.051908.125345","article-title":"Botulinum neurotoxin: A marvel of protein design","volume":"79","author":"Montal","year":"2010","journal-title":"Annu. Rev. Biochem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"116516","DOI":"10.1016\/j.jns.2019.116516","article-title":"Safety and pharmacodynamics of a novel recombinant botulinum toxin E (rBoNT-E): Results of a phase 1 study in healthy male subjects compared with abobotulinumtoxinA (Dysport\u00ae)","volume":"407","author":"Pons","year":"2019","journal-title":"J. Neurol. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"847e","DOI":"10.1097\/PRS.0000000000005029","article-title":"Safety and Efficacy of EB-001, a Novel Type E Botulinum Toxin, in Subjects with Glabellar Frown Lines: Results of a Phase 2, Randomized, Placebo-Controlled, Ascending-Dose Study","volume":"142","author":"Yoelin","year":"2018","journal-title":"Plast. Reconstr. Surg."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.toxicon.2020.02.020","article-title":"Clinical duration of action of different botulinum toxin types in humans","volume":"179","author":"Eleopra","year":"2020","journal-title":"Toxicon"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/0304-4165(93)90138-X","article-title":"Purification and characterization of the ganglioside-binding fragment of Clostridium botulinum type E neurotoxin","volume":"1156","author":"Kamata","year":"1993","journal-title":"BBA\u2014Gen. Subj."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1942","DOI":"10.1111\/j.1471-4159.2009.06298.x","article-title":"Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor","volume":"110","author":"Rummel","year":"2009","journal-title":"J. Neurochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1584","DOI":"10.1111\/j.1471-4159.2009.06093.x","article-title":"Cell entry strategy of clostridial neurotoxins","volume":"109","author":"Binz","year":"2009","journal-title":"J. Neurochem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5226","DOI":"10.1091\/mbc.e08-07-0765","article-title":"Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons","volume":"19","author":"Dong","year":"2008","journal-title":"Mol. Biol. Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1042\/BJ20130391","article-title":"Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E","volume":"453","author":"Mahrhold","year":"2013","journal-title":"Biochem. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3325","DOI":"10.1016\/j.jmb.2016.05.020","article-title":"Gangliosides of the Vertebrate Nervous System","volume":"428","author":"Schnaar","year":"2016","journal-title":"J. Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"34015","DOI":"10.1074\/jbc.M111.272054","article-title":"Unique ganglioside recognition strategies for clostridial neurotoxins","volume":"286","author":"Benson","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.toxicon.2020.02.006","article-title":"Neuronal selectivity of botulinum neurotoxins","volume":"178","author":"Poulain","year":"2020","journal-title":"Toxicon"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.jmb.2008.12.027","article-title":"Domain Organization in Clostridium botulinum Neurotoxin Type E Is Unique: Its Implication in Faster Translocation","volume":"386","author":"Kumaran","year":"2009","journal-title":"J. Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"16993","DOI":"10.1074\/jbc.M710442200","article-title":"Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics","volume":"283","author":"Wang","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Stenmark, P., Dupuy, J., Imamura, A., Kiso, M., and Stevens, R.C. (2008). Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction. PLoS Pathog., 4.","DOI":"10.2210\/pdb2vu9\/pdb"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2058","DOI":"10.1038\/ncomms3058","article-title":"Structure of dual receptor binding to botulinum neurotoxin B","volume":"4","author":"Berntsson","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1111\/j.1365-2958.2011.07682.x","article-title":"The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites","volume":"81","author":"Strotmeier","year":"2011","journal-title":"Mol. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1042\/BJ20101042","article-title":"Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner","volume":"431","author":"Strotmeier","year":"2010","journal-title":"Biochem. J."},{"key":"ref_28","first-page":"1","article-title":"Two Feet on the Membrane: Uptake of Clostridial Neurotoxins","volume":"406","author":"Rummel","year":"2017","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1107\/S2059798316013553","article-title":"Glycoblocks: A schematic three-dimensional representation for glycans and their interactions","volume":"73","author":"McNicholas","year":"2017","journal-title":"Acta Crystallogr. Sect. D Struct. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery","volume":"51","author":"Laskowski","year":"2011","journal-title":"J. Chem. Inf. Model."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1021\/jacs.6b09534","article-title":"Glycans Confer Specificity to the Recognition of Ganglioside Receptors by Botulinum Neurotoxin A","volume":"139","author":"Hamark","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"14859","DOI":"10.1021\/ja036166s","article-title":"On the value of c: Can low affinity systems be studied by isothermal titration calorimetry?","volume":"125","author":"Turnbull","year":"2003","journal-title":"J. Am. Chem. Soc."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5631","DOI":"10.1021\/bi9002138","article-title":"Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F","volume":"48","author":"Fu","year":"2009","journal-title":"Biochemistry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1038\/nsmb.3245","article-title":"N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A","volume":"23","author":"Yao","year":"2016","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1046\/j.1365-2958.2003.03872.x","article-title":"The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction","volume":"51","author":"Rummel","year":"2004","journal-title":"Mol. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"XDS","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Cryst. D Biol. Cryst."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1107\/S0907444913000061","article-title":"How good are my data and what is the resolution?","volume":"69","author":"Evans","year":"2013","journal-title":"Acta Cryst. D Biol. Cryst."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"(1994). The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr., 50, 760\u2013763.","DOI":"10.1107\/S0907444994003112"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J. Appl. Crystallogr."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1107\/S0907444911001314","article-title":"REFMAC5 for the refinement of macromolecular crystal structures","volume":"67","author":"Murshudov","year":"2011","journal-title":"Acta Cryst. D Biol. Cryst."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1038\/nsmb.3115","article-title":"Privateer: Software for the conformational validation of carbohydrate structures","volume":"22","author":"Agirre","year":"2015","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2010","journal-title":"Acta Cryst. D Biol. Cryst."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Davies, J.R., Masuyer, G., and Stenmark, P. (2020). Structural and Biochemical Characterization of Botulinum Neurotoxin Subtype B2 Binding to Its Receptors. Toxins, 12.","DOI":"10.3390\/toxins12090603"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/8315\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:39:16Z","timestamp":1760164756000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/15\/8315"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,2]]},"references-count":44,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["ijms22158315"],"URL":"https:\/\/doi.org\/10.3390\/ijms22158315","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,2]]}}}