| 1. |
- 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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| 2. |
- 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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| 3. |
- Martínez-Carranza, Markel, et al.
(författare)
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Structure and pH stability of botulinum neurotoxin X in complex with NTNH
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Prejanò, Mario, et al.
(författare)
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Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:31, s. 14258-14268
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Tidskriftsartikel (refereegranskat)abstract
- Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleo-tide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.
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| 5. |
- Škerlová, Jana, et al.
(författare)
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Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target
- 2020
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 295:33, s. 11656-11668
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Tidskriftsartikel (refereegranskat)abstract
- The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.
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| 6. |
- Škerlová, Jana, et al.
(författare)
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Structural and functional analysis of the inhibition of equine glutathione transferase A3-3 by organotin endocrine disrupting pollutants
- 2021
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Ingår i: Environmental Pollution. - : Elsevier BV. - 0269-7491 .- 1873-6424. ; 268
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Tidskriftsartikel (refereegranskat)abstract
- Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrinedisrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects.
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| 7. |
- Škerlová, Jana, et al.
(författare)
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Structure and steroid isomerase activity of Drosophila glutathione transferase E14 essential for ecdysteroid biosynthesis
- 2020
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 594:7, s. 1187-1195
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Tidskriftsartikel (refereegranskat)abstract
- Ecdysteroids are critically important for the formation of the insect exoskeleton. Cholesterol is a precursor of ecdysone and its active form 20-hydroxyecdysone, but some steps in the ecdysteroid biosynthesis pathway remain unknown. An essential requirement of glutathione (GSH) transferase GSTE14 in ecdysteroid biosynthesis has been established in Drosophila melanogaster, but its function is entirely unknown. Here, we have determined the crystal structure of GSTE14 in complex with GSH and investigated the kinetic properties of GSTE14 with alternative substrates. GSTE14 has high-ranking steroid double-bond isomerase activity, albeit 50-fold lower than the most efficient mammalian GSTs. Corresponding steroid isomerizations are unknown in insects, and their exact physiological role remains to be shown. Nonetheless, the essential enzyme GSTE14 is here demonstrated to be catalytically competent and have a steroid-binding site.
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| 8. |
- Škerlová, Jana, et al.
(författare)
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Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
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| 9. |
- Škerlová, Jana, et al.
(författare)
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Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion
- 2021
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- The pyruvate dehydrogenase complex (PDHc) is a large multienzyme complex that converts pyruvate into acetyl-coenzyme A and in E. coli the core of the PDHc is formed by 24 copies of dihydrolipoyl transacetylase. Here, the authors present the cryo-EM structure of the E. coli dihydrolipoyl transacetylase 24-mer core in a native resting state including lipoyl domains, and discuss the mechanism of substrate shuttling by the lipoyl domains. The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
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