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Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 82018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4518Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 angstrom X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.

Place, publisher, year, edition, pages
2018. Vol. 8, article id 4518
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-156106DOI: 10.1038/s41598-018-22842-4ISI: 000427366200048PubMedID: 29540745OAI: oai:DiVA.org:su-156106DiVA, id: diva2:1205160
Available from: 2018-05-11 Created: 2018-05-11 Last updated: 2018-08-27Bibliographically approved
In thesis
1. Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
Open this publication in new window or tab >>Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

X-ray crystallography visualizes the three dimensional molecular structures of proteins at atomic resolution. Seeing the molecular structure of a biomedically interesting protein enables a higher understanding of its function. The process of producing pure protein from genetic material to generate crystals and determine the molecular structure can be a long and challenging process. My thesis involves structural and functional studies of two different proteins, which are both biomedically interesting and important to learn about. X-ray crystallography is the method which has been used to determine the majority of the protein structures that we know of today and is also the method used in the results presented in my thesis. 

Today there are no cancer therapies defeating all types of cancers and they do not come without side effects. Battling cancer diseases often include long and painful treatments. Finding an anti-cancer drug targeting phenotypes characteristic of cancer cells is a compelling thought. MutT homolog-1 (MTH1) is an enzyme present in all proliferating cells. The enzyme seems to be crucial for cancer cell survival but not for the viability of normal cells. MTH1 cleans out oxidized and thereby damaged nucleotides from the free nucleotide pool and stops them from being used in DNA synthesis. This process is very important in fast proliferating cancer cells. The hypothesis is to inhibit MTH1 and thereby allow a limitless amount of DNA damage in the cancer cells. This action will eventually kill cancer cells while not affecting normal cells. The molecular structure of MTH1 with (PDB ID: 3ZR0) and without a product bound (PDB ID: 3ZR1) was determined and is presented in my thesis. These two structures aided in the synthesis of inhibitors. 

Botulinum neurotoxins (BoNTs) are the most potent toxins known. As little as one gram of pure toxin could potentially kill one million people. Due to its potency BoNT is a potential  bioterrorism threat. The toxin is also a very potent drug used clinically to relieve the symptoms of an array of neuromuscular disorders. Most people know this neurotoxin by one of its commercial names: Botox™. Additionally BoNTs are the cause of botulism. BoNTs are neuro-specific enzymes that target neuromuscular signaling, inducing flaccid paralysis and potentially death. It is of importance to learn more about these toxins to enable the development of new countermeasures, vaccines or more efficient neuroparalytic drugs. BoNTs consist of three domains with different functions, all crucial for intoxication. The toxins are fragile and can easily be destroyed by harsh surroundings if not protected by non-toxic non-hemagglutinin (NTNH) proteins. The complex of some BoNT serotypes and their protective NTNH have proven to be pH-dependent. Parts of the intoxication process are not yet clear and their mechanisms are still puzzling researchers. Until recently seven BoNT serotypes were identified. We have now identified and characterized a novel serotype called BoNT/X. The molecular structure of the active domain is presented here (PDB ID: 6F47). The pH-dependent mechanism forming a complex as seen in other serotypes, is confirmed to be present in BoNT/X as well.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 50
Keywords
Botulinum neurotoxin, botulism, cancer, gene cluster, molecular structures, MTH1, NTNH, oxidised nucleotides, progenitor complex, protein expression, protein purification, X-ray crystallography
National Category
Other Chemistry Topics
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-159295 (URN)978-91-7797-384-3 (ISBN)978-91-7797-385-0 (ISBN)
Public defence
2018-10-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2018-09-17 Created: 2018-08-26 Last updated: 2018-09-17Bibliographically approved

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