{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T20:46:47Z","timestamp":1790714807061,"version":"4.1.0"},"reference-count":49,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2017,3,8]],"date-time":"2017-03-08T00:00:00Z","timestamp":1488931200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["R01AI091823"],"award-info":[{"award-number":["R01AI091823"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["R01AI125704"],"award-info":[{"award-number":["R01AI125704"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["R21AI123920"],"award-info":[{"award-number":["R21AI123920"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT\/A\u2013G), a new mosaic toxin type termed BoNT\/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT\/HA is ~84% identical to that of BoNT\/A1. However, BoNT\/HA responds differently to some potent BoNT\/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT\/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT\/HA-HC at 1.8 \u00c5. Sequence and structure analyses reveal that BoNT\/HA and BoNT\/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT\/HA compared to BoNT\/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.<\/jats:p>","DOI":"10.3390\/toxins9030093","type":"journal-article","created":{"date-parts":[[2017,3,8]],"date-time":"2017-03-08T07:42:58Z","timestamp":1488958978000},"page":"93","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H"],"prefix":"10.3390","volume":"9","author":[{"given":"Guorui","family":"Yao","sequence":"first","affiliation":[{"name":"Department of Physiology &amp; Biophysics, University of California, Irvine, CA 92697, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kwok-ho","family":"Lam","sequence":"additional","affiliation":[{"name":"Department of Physiology &amp; Biophysics, University of California, Irvine, CA 92697, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4046-1704","authenticated-orcid":false,"given":"Kay","family":"Perry","sequence":"additional","affiliation":[{"name":"NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6299-9257","authenticated-orcid":false,"given":"Jasmin","family":"Weisemann","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0645-1641","authenticated-orcid":false,"given":"Andreas","family":"Rummel","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rongsheng","family":"Jin","sequence":"additional","affiliation":[{"name":"Department of Physiology &amp; Biophysics, University of California, Irvine, CA 92697, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,3,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Botulinum toxin as a biological weapon: Medical and public health management","volume":"285","author":"Arnon","year":"2001","journal-title":"JAMA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/j.tox.2005.06.015","article-title":"Medical aspects of toxin weapons","volume":"214","author":"Bigalke","year":"2005","journal-title":"Toxicology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1093\/infdis\/154.2.207","article-title":"Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy","volume":"154","author":"Aureli","year":"1986","journal-title":"J. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/S0072-9752(07)01511-4","article-title":"Botulism","volume":"91","author":"Johnson","year":"2008","journal-title":"Handb. Clin. Neurol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"832","DOI":"10.1038\/359832a0","article-title":"Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin","volume":"359","author":"Schiavo","year":"1992","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1038\/365160a0","article-title":"Botulinum neurotoxin a selectively cleaves the synaptic protein SNAP-25","volume":"365","author":"Blasi","year":"1993","journal-title":"Nature"},{"key":"ref_7","first-page":"21","article-title":"Assembly and function of the botulinum neurotoxin progenitor complex","volume":"364","author":"Gu","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1016\/j.bbrc.2014.03.008","article-title":"High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex","volume":"446","author":"Lee","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1126\/science.1253823","article-title":"Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex","volume":"344","author":"Lee","year":"2014","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1046\/j.1365-2672.1997.00313.x","article-title":"Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins","volume":"84","author":"Collins","year":"1998","journal-title":"J. Appl. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1126\/science.1214270","article-title":"Botulinum neurotoxin is shielded by NTNHA in an interlocked complex","volume":"335","author":"Gu","year":"2012","journal-title":"Science"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Lee, K., Gu, S., Jin, L., Le, T.T., Cheng, L.W., Strotmeier, J., Kruel, A.M., Yao, G., Perry, K., and Rummel, A. (2013). Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003690"},{"key":"ref_13","first-page":"1","article-title":"Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes","volume":"364","author":"Hill","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/nrmicro3295","article-title":"Botulinum neurotoxins: Genetic, structural and mechanistic insights","volume":"12","author":"Rossetto","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"38","DOI":"10.3390\/toxins9010038","article-title":"Historical perspectives and guidelines for botulinum neurotoxin subtype nomenclature","volume":"9","author":"Peck","year":"2017","journal-title":"Toxins"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/0167-4781(96)00006-1","article-title":"Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms","volume":"1307","author":"Moriishi","year":"1996","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1602","DOI":"10.1016\/j.str.2013.06.026","article-title":"Crystal structures of botulinum neurotoxin DC in complex with its protein receptors synaptotagmin I and II","volume":"21","author":"Berntsson","year":"2013","journal-title":"Structure"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1093\/infdis\/jit449","article-title":"A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins","volume":"209","author":"Barash","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1093\/infdis\/jit450","article-title":"Molecular characterization of a novel botulinum neurotoxin type H gene","volume":"209","author":"Dover","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1093\/infdis\/jiv327","article-title":"A novel botulinum neurotoxin, previously reported as serotype H, has a hybrid-like structure with regions of similarity to the structures of serotypes A and F and is neutralized with serotype A antitoxin","volume":"213","author":"Maslanka","year":"2016","journal-title":"J. Infect. Dis."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Pellett, S., Tepp, W.H., Bradshaw, M., Kalb, S.R., Dykes, J.K., Lin, G., Nawrocki, E.M., Pier, C.L., Barr, J.R., and Maslanka, S.E. (2016). Purification and characterization of botulinum neurotoxin FA from a genetically modified Clostridium botulinum strain. mSphere, 1.","DOI":"10.1128\/mSphere.00100-15"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1606","DOI":"10.1093\/infdis\/jiv770","article-title":"Immunological characterization and neutralizing ability of monoclonal antibodies directed against botulinum neurotoxin type H","volume":"213","author":"Fan","year":"2016","journal-title":"J. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Gonzalez-Escalona, N., Thirunavukkarasu, N., Singh, A., Toro, M., Brown, E.W., Zink, D., Rummel, A., and Sharma, S.K. (2014). Draft genome sequence of bivalent Clostridium botulinum strain IBCA10\u20137060, encoding botulinum neurotoxin B and a new FA mosaic type. Genome Announc., 2.","DOI":"10.1128\/genomeA.01275-14"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.febslet.2011.11.033","article-title":"Discovery of a novel enzymatic cleavage site for botulinum neurotoxin F5","volume":"586","author":"Kalb","year":"2012","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"12764","DOI":"10.1016\/S0021-9258(18)99941-2","article-title":"Cleavage of members of the synaptobrevin\/vamp family by types D and F botulinal neurotoxins and tetanus toxin","volume":"269","author":"Yamasaki","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_26","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_27","first-page":"3233","article-title":"Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins","volume":"125","author":"Peng","year":"2012","journal-title":"J. Cell Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1016\/0968-0004(86)90282-3","article-title":"How do tetanus and botulinum toxins bind to neuronal membranes","volume":"11","author":"Montecucco","year":"1986","journal-title":"Trends Biochem. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Kull, S., Schulz, K.M., Weisemann, J., Kirchner, S., Schreiber, T., Bollenbach, A., Dabrowski, P.W., Nitsche, A., Kalb, S.R., and Dorner, M.B. (2015). Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0116381"},{"key":"ref_30","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_31","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1073\/pnas.0609713104","article-title":"Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept","volume":"104","author":"Rummel","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1016\/j.febslet.2014.02.034","article-title":"Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A","volume":"588","author":"Strotmeier","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Rummel, A. (2016). Two feet on the membrane: Uptake of clostridial neurotoxins. Curr. Top. Microbiol. Immunol.","DOI":"10.1007\/82_2016_48"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1038\/nature12732","article-title":"Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A","volume":"505","author":"Benoit","year":"2014","journal-title":"Nature"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1016\/S0306-4522(99)00370-X","article-title":"SV2C is a synaptic vesicle protein with an unusually restricted localization: Anatomy of a synaptic vesicle protein family","volume":"94","author":"Janz","year":"1999","journal-title":"Neuroscience"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.brainres.2010.09.063","article-title":"Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system","volume":"1367","author":"Dardou","year":"2011","journal-title":"Brain Res."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Crevecoeur, J., Foerch, P., Doupagne, M., Thielen, C., Vandenplas, C., Moonen, G., Deprez, M., and Rogister, B. (2013). Expression of SV2 isoforms during rodent brain development. BMC Neurosci., 14.","DOI":"10.1186\/1471-2202-14-87"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1093\/toxsci\/kfr354","article-title":"Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection","volume":"126","author":"Whitemarsh","year":"2012","journal-title":"Toxicol. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5047","DOI":"10.1128\/AAC.02830-14","article-title":"Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A","volume":"58","author":"Nayak","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.jmb.2006.09.084","article-title":"Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display","volume":"365","author":"Levy","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.bios.2009.06.031","article-title":"Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies","volume":"25","author":"Warner","year":"2009","journal-title":"Biosens. Bioelectron."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/nbt1269","article-title":"Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin","volume":"25","author":"Levy","year":"2007","journal-title":"Nat. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1107\/S0907444910048675","article-title":"Imosflm: A new graphical interface for diffraction-image processing with mosflm","volume":"67","author":"Battye","year":"2011","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_45","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_46","doi-asserted-by":"crossref","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: Model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.1107\/S0907444903008126","article-title":"A graphical user interface to the CCP4 program suite","volume":"59","author":"Potterton","year":"2003","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1038\/355472a0","article-title":"Free R value: A novel statistical quantity for assessing the accuracy of crystal structures","volume":"355","author":"Brunger","year":"1992","journal-title":"Nature"},{"key":"ref_49","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 Crystallogr. D Biol. Crystallogr."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/3\/93\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:30:00Z","timestamp":1760207400000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/3\/93"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,3,8]]},"references-count":49,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2017,3]]}},"alternative-id":["toxins9030093"],"URL":"https:\/\/doi.org\/10.3390\/toxins9030093","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,3,8]]}}}