Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-0192-9762
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 [en]
botulinum neurotoxin
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-178474ISBN: 978-91-7911-002-4 (print)ISBN: 978-91-7911-003-1 (electronic)OAI: oai:DiVA.org:su-178474DiVA, id: diva2:1389357
Public defence
2020-03-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
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
List of papers
1. Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster
Open this publication in new window or tab >>Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster
Show others...
2017 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 22, p. 3781-3792Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

Keywords
botulinum neurotoxin, Crystallography, gene cluster, TULIP, X-ray
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-150675 (URN)10.1002/1873-3468.12889 (DOI)000416726600008 ()
Funder
Swedish Research Council, 2014-5667Wenner-Gren FoundationsSwedish Cancer Society
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2020-01-29Bibliographically approved
2. Synaptotagmin Binding to Botulinum Neurotoxins
Open this publication in new window or tab >>Synaptotagmin Binding to Botulinum Neurotoxins
Show others...
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.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-178174 (URN)10.1021/acs.biochem.9b00554 (DOI)000511509600017 ()
Available from: 2020-01-20 Created: 2020-01-20 Last updated: 2020-03-02Bibliographically approved
3. Identification and characterization of a novel botulinum neurotoxin
Open this publication in new window or tab >>Identification and characterization of a novel botulinum neurotoxin
Show others...
2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 14130Article in journal (Refereed) Published
Abstract [en]

Botulinum neurotoxins are known to have seven serotypes (BoNT/A-G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-145842 (URN)10.1038/ncomms14130 (DOI)000406846300001 ()
Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2020-02-20Bibliographically approved
4. Structure and pH stability of botulinum neurotoxin X in complex with NTNH
Open this publication in new window or tab >>Structure and pH stability of botulinum neurotoxin X in complex with NTNH
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Botulinum neurotoxins (BoNTs) are the most potent toxins known to man and are also used to treat an increasing number of medical disorders. They target the neuromuscular junction and inhibit synaptic vesicle exocytosis in motor neurons, thereby causing paralysis. The molecular architecture of BoNTs comprises the receptor-binding domain, translocation domain, and zinc dependent protease domain. BoNTs are naturally co-expressed with a non-toxic non-hemagglutinin partner (NTNH) with which they form the minimal progenitor toxin complex to resist the low pH and proteases in the intestine, before they cross the intestinal barrier in the host. The full-length structures of BoNT/A, BoNT/B and BoNT/E have been determined and the structures of minimal progenitor toxin complexes of BoNT/A and BoNT/E are also available.

We have recently identified and characterized a new botulinum neurotoxin serotype, BoNT/X. It shares the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. BoNT/X cleaves its substrates at a novel site and is the only BoNT that also cleaves other non-canonical substrates. The only structural information currently available for this novel toxin is the structure of its protease domain (light chain).

We have determined the structure of the 300 kDa BoNT/X-NTNH complex at 3.12 Å resolution using single-particle cryo-electron microscopy. This structure together with the pH stability analysis of the complex provides the molecular basis to understand the toxin’s interactions with its protective partner and also the evolutionary relationships between BoNT serotypes.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-178180 (URN)
Available from: 2020-01-20 Created: 2020-01-20 Last updated: 2020-02-20Bibliographically approved
5. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
Open this publication in new window or tab >>The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
Show others...
2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 4403Article in journal (Refereed) Published
Abstract [en]

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175701 (URN)10.1038/s41467-019-12301-7 (DOI)000488232600011 ()31562305 (PubMedID)
Available from: 2019-11-13 Created: 2019-11-13 Last updated: 2020-01-29Bibliographically approved

Open Access in DiVA

Structural Insights into Botulinum Neurotoxins and the ALFA-tag System(38264 kB)15 downloads
File information
File name FULLTEXT01.pdfFile size 38264 kBChecksum SHA-512
a60a3ff47a119cd2c3156a318a6f78442a7f98f7bff9ce3bf24ce3d4cf8033349906935951cbadc6fa449e83299147a7454916857cd486c6964a49cceb67234d
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Martínez-Carranza, Markel
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 15 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 338 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf