| 1. |
- Kinsolving, Julia, et al.
(författare)
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Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7
- 2024
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Ingår i: Cell Reports. - 2211-1247. ; 43:2
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Tidskriftsartikel (refereegranskat)abstract
- The G protein -coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live -cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA -approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore -forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
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| 2. |
- Kosenina, Sara, 1993-, et al.
(författare)
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Crystal structure of the catalytic domain of BoNT/X in complex with its substrate, VAMP2
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Botulinum neurotoxins (BoNTs) are the deadliest toxins known to man but are at the same time being used for the treatment of an increasing number of medical conditions. BoNTs are zinc proteases that act in presynaptic cholinergic motoneurons by cleaving SNARE proteins, hence inhibiting neurotransmission. Structural information on the BoNT catalytic domain is crucial for a detailed understanding of their substrate specificity and binding mechanism.Here we report the 1.85 Å crystal structure of the catalytic light chain of BoNT/X in complex with its substrate, VAMP2. The structure sheds light on the sites important for VAMP2 binding and will help in the engineering of novel therapeutic toxins with altered and improved substrate specificity.
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| 3. |
- Košenina, Sara, et al.
(författare)
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Crystal structure of the catalytic domain of the Weissella oryzae botulinum-like toxin
- 2019
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 593:12, s. 1403-1410
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins (BoNTs) are the most potent toxins known. So far, eight serotypes have been identified that all act as zinc-dependent endopeptidases targeting SNARE proteins and inhibiting the release of neurotransmitters. Recently, the first botulinum toxin-like protein was identified outside the Clostridial genus, designated BoNT/Wo in the genome of Weissella oryzae. Here, we report the 1.6 angstrom X-ray crystal structure of the light chain of BoNT/Wo (LC/Wo). LC/Wo presents the core fold common to BoNTs but has an unusually wide, open and negatively charged catalytic pocket, with an additional Ca2+ ion besides the zinc ion and a unique ss-hairpin motif. The structural information will help establish the substrate profile of BoNT/Wo and help our understanding of how BoNT evolved.
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| 4. |
- Košenina, Sara, et al.
(författare)
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Crystal structure of the OrfX1–OrfX3 complex from the PMP1 neurotoxin gene cluster
- 2023
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 597:4, s. 515-523
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Tidskriftsartikel (refereegranskat)abstract
- Paraclostridial mosquitocidal protein 1 (PMP1) is a member of the clostridial neurotoxin (CNT) family, which includes botulinum and tetanus neurotoxins. PMP1 has unique selectivity for anopheline mosquitos and is the only known member of the family that targets insects. PMP1 is encoded in an orfX gene cluster, which in addition to the toxin, consists of OrfX1, OrfX2, OrfX3, P47 and NTNH, which have been shown to aid in PMP1 toxicity. We here show that OrfX1 and OrfX3 form a complex and present its structure at 2.7 Å. The OrfX1–OrfX3 complex mimics the structure of full-length OrfX2 and belongs to the lipid-binding TULIP protein superfamily. With this report, the structures of all proteins encoded in the orfX gene cluster of CNTs are now determined.
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| 5. |
- Kosenina, Sara, et al.
(författare)
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Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM
- 2022
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Ingår i: Toxins. - : MDPI AG. - 2072-6651. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.
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| 6. |
- Košenina, Sara, 1993-, et al.
(författare)
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The cryo-EM structure of the BoNT/Wo-NTNH complex reveals two immunoglobulin-like domains
- 2024
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Ingår i: The FEBS Journal. - 1742-464X .- 1742-4658. ; 291:4, s. 676-689
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Tidskriftsartikel (refereegranskat)abstract
- The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like (Big) domains on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT–NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.
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| 7. |
- Kosenina, Sara, et al.
(författare)
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The cryo-EM structure of the BoNT/Wo NTNH complex reveals two immunoglobulin-like domains
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin’s interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like domains (designated Big domains) on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT–NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates. Both the BoNT/Wo and the NTNH/Wo have several unique structural features not seen in traditional BoNTs, including two bacterial immunoglobulin-like domains as parts of the NTNH/Wo that may contribute to receptor recognition during BoNT/Wo intoxication.
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| 8. |
- Luttens, Andreas, et al.
(författare)
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Virtual Fragment Screening for DNA Repair Inhibitors in Vast Chemical Space
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging hit optimization step. In efforts to identify DNA repair inhibitors, we explored the use of structure-based virtual screening to access ultralarge fragment libraries that cover four orders of magnitude larger fractions of chemical space than traditional techniques. A set of 14 million fragments were docked to 8-oxoguanine DNA glycosylase (OGG1), a challenging drug target involved in cancer and inflammation. Of the 29 top-ranked fragments that were experimentally evaluated, four compounds were shown to bind to OGG1 and X-ray crystallography confirmed the predicted binding modes. Docking of readily synthesizable elaborations guided fragment optimization, leading to the discovery of submicromolar OGG1 inhibitors with anti-inflammatory and anti-cancer effects in cell models. Our results demonstrate that fragment-based virtual screening enables efficient exploration of vast chemical libraries.
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| 9. |
- Masuyer, Geoffrey, et al.
(författare)
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Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Persson Košenina, Sara, 1993-
(författare)
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Botulinum neurotoxins
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Botulinum neurotoxins (BoNTs) are the most potent toxins known to man, with less than 1 μg of pure toxin being enough to kill an adult man. Despite the high toxicity, BoNTs are widely used in cosmetics and in medicine for the treatment of an increasing number of medical conditions.BoNTs have a conserved structure that consists of three domains (a receptor binding, translocation, and catalytic domain). The receptor binding domain is responsible for binding to neuronal receptors, the translocation domain is a delivery vehicle that transports the catalytic domain into the cytosol, where the latter cleaves its target - proteins of the SNARE family, inhibiting neurotransmitter release and consequently causing muscle paralysis.BoNTs are produced by the bacteria Clostridium botulinum together with several other accessory proteins, which are responsible for shielding BoNTs in the harsh environment of the target gastrointestinal tract and assisting them in crossing the epithelial barrier between the gastrointestinal tract and general circulation.Several BoNT serotypes (A-G) have been identified over the years. Additionally, several BoNT-like toxins have been identified in non-Clostridial types of bacteria. Namely, these proteins are BoNT/Wo, BoNT/En and PMP1.In this thesis, we present six papers, where we studied both the canonical BoNTs and the new BoNT-like toxins as well as their accessory proteins using structural biology techniques, such as X-ray crystallography and cryo-EM. Elucidating the structures of these proteins is crucial for understanding their function and mechanism of action.
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