Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Lam, Kwok-ho; Tremblay, Jacqueline M.; Perry, Kay; Ichtchenko, Konstantin; Shoemaker, Charles B.; Jin, Rongsheng; McClane, ed., Bruce A.

doi:10.1371/journal.ppat.1010169

Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Journal Article · Thu Jan 06 00:00:00 EST 2022 · PLoS Pathogens

DOI:https://doi.org/10.1371/journal.ppat.1010169· OSTI ID:1841040

; Tremblay, Jacqueline M.; ; ; ; ; McClane, ed., Bruce A.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE; USDOE Office of Science (SC)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1841040

Alternate ID(s):: OSTI ID: 1838551
OSTI ID: 2470176

Journal Information:: PLoS Pathogens, Journal Name: PLoS Pathogens Journal Issue: 1 Vol. 18; ISSN 1553-7374

Publisher:: Public Library of Science (PLoS)Copyright Statement

Country of Publication:: United States

Language:: English

References (47)

Shape Complementarity at Protein/Protein Interfaces Lawrence, Michael C.; Colman, Peter M. Journal of Molecular Biology, Vol. 234, Issue 4 https://doi.org/10.1006/jmbi.1993.1648	journal	December 1993
Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography Kumaran, Desigan; Adler, Michael; Levit, Matthew Bioorganic & Medicinal Chemistry, Vol. 23, Issue 22 https://doi.org/10.1016/j.bmc.2015.10.024	journal	November 2015
Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins Lam, Kwok-ho; Tremblay, Jacqueline M.; Vazquez-Cintron, Edwin Cell Reports, Vol. 30, Issue 8 https://doi.org/10.1016/j.celrep.2020.01.107	journal	February 2020
Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility Silvaggi, Nicholas R.; Boldt, Grant E.; Hixon, Mark S. Chemistry & Biology, Vol. 14, Issue 5 https://doi.org/10.1016/j.chembiol.2007.03.014	journal	May 2007
Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein, Cp-P34, unique to Cryptosporidium Jaskiewicz, Justyna J.; Tremblay, Jacqueline M.; Tzipori, Saul International Journal for Parasitology, Vol. 51, Issue 9 https://doi.org/10.1016/j.ijpara.2021.01.003	journal	August 2021
Identification of the Amino Acid Residues Rendering TI-VAMP Insensitive toward Botulinum Neurotoxin B Sikorra, Stefan; Henke, Tina; Swaminathan, Subramanyam Journal of Molecular Biology, Vol. 357, Issue 2 https://doi.org/10.1016/j.jmb.2005.12.075	journal	March 2006
A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic α-Exosite Binding Region Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng Journal of Molecular Biology, Vol. 397, Issue 4 https://doi.org/10.1016/j.jmb.2010.01.070	journal	April 2010
The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability Baldwin, Michael R.; Bradshaw, Marite; Johnson, Eric A. Protein Expression and Purification, Vol. 37, Issue 1 https://doi.org/10.1016/j.pep.2004.05.009	journal	September 2004
Camelid nanobodies: killing two birds with one stone Desmyter, Aline; Spinelli, Silvia; Roussel, Alain Current Opinion in Structural Biology, Vol. 32 https://doi.org/10.1016/j.sbi.2015.01.001	journal	June 2015
A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate Zuniga, Jorge E.; Schmidt, James J.; Fenn, Timothy Structure, Vol. 16, Issue 10 https://doi.org/10.1016/j.str.2008.07.011	journal	October 2008
Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases Tremblay, Jacqueline M.; Kuo, Chueh-Ling; Abeijon, Claudia Toxicon, Vol. 56, Issue 6 https://doi.org/10.1016/j.toxicon.2010.07.003	journal	November 2010
Insights into the Different Catalytic Activities of Clostridium Neurotoxins Chen, Sheng; Karalewitz, Andrew P. A.; Barbieri, Joseph T. Biochemistry, Vol. 51, Issue 18 https://doi.org/10.1021/bi3000098	journal	April 2012
Crystal structure of botulinum neurotoxin type A and implications for toxicity Lacy, D. Borden; Tepp, William; Cohen, Alona C. Nature Structural Biology, Vol. 5, Issue 10 https://doi.org/10.1038/2338	journal	October 1998
Free R value: a novel statistical quantity for assessing the accuracy of crystal structures Brünger, Axel T. Nature, Vol. 355, Issue 6359 https://doi.org/10.1038/355472a0	journal	January 1992
Substrate recognition strategy for botulinum neurotoxin serotype A Breidenbach, Mark A.; Brunger, Axel T. Nature, Vol. 432, Issue 7019 https://doi.org/10.1038/nature03123	journal	December 2004
Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin Garcia-Rodriguez, Consuelo; Levy, Raphael; Arndt, Joseph W. Nature Biotechnology, Vol. 25, Issue 1 https://doi.org/10.1038/nbt1269	journal	December 2006
A general protocol for the generation of Nanobodies for structural biology Pardon, Els; Laeremans, Toon; Triest, Sarah Nature Protocols, Vol. 9, Issue 3 https://doi.org/10.1038/nprot.2014.039	journal	February 2014
Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F Agarwal, Rakhi; Schmidt, James J.; Stafford, Robert G. Nature Structural & Molecular Biology, Vol. 16, Issue 7 https://doi.org/10.1038/nsmb.1626	journal	June 2009
A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-07457-5	journal	August 2017
A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin Feltrup, Thomas M.; Patel, Kruti; Kumar, Raj Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-018-26764-z	journal	June 2018
Plasma membrane localization signals in the light chain of botulinum neurotoxin Fernandez-Salas, E.; Steward, L. E.; Ho, H. Proceedings of the National Academy of Sciences, Vol. 101, Issue 9 https://doi.org/10.1073/pnas.0400229101	journal	February 2004
A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis Wang, Jiafu; Zurawski, Tomas H.; Meng, Jianghui Journal of Biological Chemistry, Vol. 286, Issue 8 https://doi.org/10.1074/jbc.M110.181784	journal	February 2011
The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site Mizanur, Rahman M.; Frasca, Verna; Swaminathan, Subramanyam Journal of Biological Chemistry, Vol. 288, Issue 33 https://doi.org/10.1074/jbc.M113.451286	journal	August 2013
Mechanism of Substrate Recognition by Botulinum Neurotoxin Serotype A Chen, Sheng; Kim, Jung-Ja P.; Barbieri, Joseph T. Journal of Biological Chemistry, Vol. 282, Issue 13 https://doi.org/10.1074/jbc.M611211200	journal	March 2007
Substrate Recognition Mechanism of VAMP/Synaptobrevin-cleaving Clostridial Neurotoxins Sikorra, Stefan; Henke, Tina; Galli, Thierry Journal of Biological Chemistry, Vol. 283, Issue 30 https://doi.org/10.1074/jbc.M800610200	journal	July 2008
Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin Chen, Sheng; Hall, Cherisse; Barbieri, Joseph T. Journal of Biological Chemistry, Vol. 283, Issue 30 https://doi.org/10.1074/jbc.M800611200	journal	May 2008
Shape complementarity and hydrogen bond preferences in protein–protein interfaces: implications for antibody modeling and protein–protein docking Kuroda, Daisuke; Gray, Jeffrey J. Bioinformatics, Vol. 32, Issue 16 https://doi.org/10.1093/bioinformatics/btw197	journal	April 2016
Potent enzyme inhibitors derived from dromedary heavy-chain antibodies Lauwereys, M. The EMBO Journal, Vol. 17, Issue 13 https://doi.org/10.1093/emboj/17.13.3512	journal	July 1998
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
MolProbity : all-atom structure validation for macromolecular crystallography Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1 https://doi.org/10.1107/S0907444909042073	journal	December 2009
XDS Kabsch, Wolfgang Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2 https://doi.org/10.1107/S0907444909047337	journal	January 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Overview of the CCP 4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910045749	journal	March 2011
Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex Gu, S.; Rumpel, S.; Zhou, J. Science, Vol. 335, Issue 6071 https://doi.org/10.1126/science.1214270	journal	February 2012
Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism Miyashita, Shin-Ichiro; Zhang, Jie; Zhang, Sicai Science Translational Medicine, Vol. 13, Issue 575 https://doi.org/10.1126/scitranslmed.aaz4197	journal	January 2021
Neuronal delivery of antibodies has therapeutic effects in animal models of botulism McNutt, Patrick M.; Vazquez-Cintron, Edwin J.; Tenezaca, Luis Science Translational Medicine, Vol. 13, Issue 575 https://doi.org/10.1126/scitranslmed.abd7789	journal	January 2021
Safety and Pharmacokinetics of XOMA 3AB, a Novel Mixture of Three Monoclonal Antibodies against Botulinum Toxin A Nayak, S. U.; Griffiss, J. M.; McKenzie, R. Antimicrobial Agents and Chemotherapy, Vol. 58, Issue 9 https://doi.org/10.1128/AAC.02830-14	journal	June 2014
Nanobodies to Study G Protein–Coupled Receptor Structure and Function Manglik, Aashish; Kobilka, Brian K.; Steyaert, Jan Annual Review of Pharmacology and Toxicology, Vol. 57, Issue 1 https://doi.org/10.1146/annurev-pharmtox-010716-104710	journal	January 2017
A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model Mukherjee, Jean; Tremblay, Jacqueline M.; Leysath, Clinton E. PLoS ONE, Vol. 7, Issue 1 https://doi.org/10.1371/journal.pone.0029941	journal	January 2012
Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity Fan, Yongfeng; Geren, Isin N.; Dong, Jianbo PLOS ONE, Vol. 10, Issue 8 https://doi.org/10.1371/journal.pone.0135306	journal	August 2015
A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong PLOS ONE, Vol. 12, Issue 3 https://doi.org/10.1371/journal.pone.0174187	journal	March 2017
Botulinum Neurotoxin Heavy Chain Belt as an Intramolecular Chaperone for the Light Chain Brunger, Axel T.; Breidenbach, Mark A.; Jin, Rongsheng PLoS Pathogens, Vol. 3, Issue 9 https://doi.org/10.1371/journal.ppat.0030113	journal	January 2007
Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A Kumaran, Desigan; Rawat, Richa; Ahmed, S. Ashraf PLoS Pathogens, Vol. 4, Issue 9 https://doi.org/10.1371/journal.ppat.1000165	journal	September 2008
A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes Garcia-Rodriguez, Consuelo; Razai, Ali; Geren, Isin Toxins, Vol. 10, Issue 3 https://doi.org/10.3390/toxins10030105	journal	March 2018
Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model Tomic, Milan; Espinoza, Yero; Martinez, Zachary Toxins, Vol. 11, Issue 4 https://doi.org/10.3390/toxins11040208	journal	April 2019
Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function Tremblay, Jacqueline M.; Vazquez-Cintron, Edwin; Lam, Kwok-Ho Toxins, Vol. 12, Issue 10 https://doi.org/10.3390/toxins12100611	journal	September 2020

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Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

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