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Synaptotagmin Binding to Botulinum Neurotoxins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-0192-9762
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2020 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 59, no 4, p. 491-498Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt-II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide’s toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.

Place, publisher, year, edition, pages
2020. Vol. 59, no 4, p. 491-498
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-178174DOI: 10.1021/acs.biochem.9b00554ISI: 000511509600017OAI: oai:DiVA.org:su-178174DiVA, id: diva2:1387094
Available from: 2020-01-20 Created: 2020-01-20 Last updated: 2020-03-02Bibliographically approved
In thesis
1. Structural Insights into Botulinum Neurotoxins and the ALFA-tag System: Structural and Functional Studies of Proteins Related to the Botulinum Neurotoxins and Design of a Novel Epitope Tag
Open this publication in new window or tab >>Structural Insights into Botulinum Neurotoxins and the ALFA-tag System: Structural and Functional Studies of Proteins Related to the Botulinum Neurotoxins and Design of a Novel Epitope Tag
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is divided into two sections; the first part describes our work in the field of botulinum neurotoxins (presented in papers I, II, III, and manuscript IV) and the second part summarizes our work involving the design of a new biochemical tool (presented in paper V).

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to date. They have a conserved structure that consists of three domains (receptor-binding, translocation, and catalytic domain), each of which has a distinct function. The receptor-binding domain binds to neuronal receptors, and after endocytosis the translocation domain shuttles the catalytic domain into the cytosol, where it cleaves neuronal proteins of the SNARE family, which are part of the vesicle-membrane fusion machinery.

In paper I, we studied proteins of unknown function (OrfX1, OrfX2, OrfX3, and P47), which are co-expressed with certain BoNTs. We solved the crystal structures of OrfX2 and P47, and their structural resemblance to tubular lipid binding proteins (TULIP) together with lipid binding studies, led us to conclude that OrfX1 and P47 are able to bind phosphatidyl inositol phosphates (PIPs) in vitro.

In paper II, we studied the binding of BoNT/B, /DC and /G to their protein receptor synaptotagmin (Syt). We determined their affinities to synaptotagmins from different species, and concluded that residue F50 in bovine Syt-II is responsible for its increased affinity towards BoNT/DC. In addition, we studied the interaction between BoNT/G and Syt-II via STD-NMR. Our results showed the binding to be similar to BoNT/B and Syt-II, and that the N-terminal region of the Syt peptide is important for the binding of BoNTs to synaptotagmin, even though it is not part of the binding interface.

In paper III and manuscript IV, we present the identification of a novel BoNT serotype named BoNT/X. We showed that BoNT/X cleaves the non-canonical substrates VAMP4, VAMP5 and Ykt6, as well as the canonical substrate VAMP1-3 at a new cleavage site, distinct from other BoNTs. In addition, we present the cryo-EM structure of BoNT/X in complex with its non-toxic interaction partner NTNH. Our pH stability experiments revealed that BoNT/X-NTNH remain bound at neutral to moderately high pH, in contrast with what is observed for BoNT/A-NTNH.

In paper V we present the design of a novel epitope tag named the ALFA system. The ALFA tag is a short α-helical protein tag that is highly stable and electroneutral. The ALFA nanobody has a very high affinity for the tag and is small enough to allow for high performance in high-resolution microscopy. The crystal structure of the ALFA nanobody in complex with the tag led to a modified version of the ALFA nanobody that can release the tag via competitive elution with free ALFA peptide. Our results showed that this system outperforms several commercially available systems in protein purification and high-resolution microscopy.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 60
Keywords
botulinum neurotoxin
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-178474 (URN)978-91-7911-002-4 (ISBN)978-91-7911-003-1 (ISBN)
Public defence
2020-03-13, 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: 2020-02-19 Created: 2020-01-29 Last updated: 2020-02-07Bibliographically approved

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