| 1. |
- Berg, Alexandra, et al.
(författare)
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Nematocida displodere mechanosensitive ion channel of small conductance 2 assembles into a unique 6-channel super-structure in vitro
- 2024
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 19:7
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Tidskriftsartikel (refereegranskat)abstract
- Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein.
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| 2. |
- Berntsson, Ronnie P. -A., et al.
(författare)
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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2725-2735
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.
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| 3. |
- Berntsson, Ronnie P. A., et al.
(författare)
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Structure of dual receptor binding to botulinum neurotoxin B
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4, s. 2058-
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-angstrom structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.
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| 4. |
- Berntsson, Ronnie P., et al.
(författare)
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Crystal Structures of Botulinum Neurotoxin DC in Complex with Its Protein Receptors Synaptotagmin I and II
- 2013
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21:9, s. 1602-1611
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs.
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| 5. |
- Breidenstein, Annika, et al.
(författare)
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PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs
- 2024
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Ingår i: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 7:8
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Tidskriftsartikel (refereegranskat)abstract
- A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.
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| 6. |
- Breidenstein, Annika, et al.
(författare)
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Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing
- 2023
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:4
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Tidskriftsartikel (refereegranskat)abstract
- Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.
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| 7. |
- Carreras-Puigvert, Jordi, et al.
(författare)
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A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.
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| 8. |
- Dunny, Gary, et al.
(författare)
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Enterococcal sex pheromones : evolutionary pathways to complex, two-signal systems
- 2016
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 198:11, s. 1556-1562
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Forskningsöversikt (refereegranskat)abstract
- Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.
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| 9. |
- Ehrenbolger, Kai Patric, 1991-
(författare)
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Nanoscopic adventures : unraveling macromolecular complexes in infectious diseases via integrative structural biology
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on understanding the underlying molecular mechanisms of infectious diseases, which claim nearly 9 million lives annually. The research centers on critical analysis of pathogen mechanisms and drug resistance. I have mainly focused on two clades of pathogens: Enterococcus faecalis and microsporidia. E. faecalis is a key nosocomial opportunistic pathogen, and microsporidia are a group of emerging fungal pathogens that considerably impact the environment and economy, causing, among other things, the decline of honeybee populations. In this thesis, I have combined biochemistry and cryo-electron microscopy to perform an in-depth molecular analysis of crucial protein complexes that drive the infectivity of these organisms. In E. faecalis, the primary drug efflux pump, EfrCD, is examined to gain insight into its role in antibiotic resistance. Microsporidia often have a drastically reduced genome and display altered macromolecular structures due to their parasitic lifestyle. The research aims to provide insights into the regulation of translational processes in microsporidia by comparing the dormant spore stage to the active intracellular stage and looking closely into the infection mechanism. In the publication “Deep mutational scan of a drug efflux pump reveals its structure– function landscape,” I determined the structure of EfrCD and several of its conformations to understand better how this protein complex contributes to E. faecalis' multidrug resistance. In further research, our focus moved to Microsporidia. During our work on the “Structure of the reduced microsporidian proteasome bound by PI31-like peptides in dormant spores” and “Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome,” we solved the structure of the endogenous microsporidian ribosome as well as multiple versions of the proteasome; the dormant form of 20S proteasome and the active form of the 20S and 26S proteasome. This gave a deeper understanding of how microsporidia could highly reduce those conserved macromolecular complexes. By discovering novel inhibitors, we were also able to understand how those energy- demanding molecular machines can efficiently regulate themselves. Furthermore, we investigated the specialized infection organ of microsporidia, known as the polar tube. As part of the paper titled "Ribosome clustering and surface layer reorganization in the microsporidian host-invasion apparatus," I contributed by performing proteomic analysis of the endogenously affinity-purified polar tubes using a native affinity tag I discovered. Additionally, I identified potential protein-protein interactions of the polar tube proteins. This complemented the work performed on the dynamics and ultrastructure remodeling of the polar tube during germination through light microscopy and cryo-electron tomography. We observed a cargo-filled state with organized arrays of ribosomes clustered along the thin tube wall and an empty post-translocation state with a thicker wall. The findings of this thesis work expand our understanding of pathogen biology and open up new possibilities for addressing drug development and drug resistance, a significant global health challenge.
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| 10. |
- Espaillat, Akbar, master, 1988-, et al.
(författare)
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A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDTGo, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDTGo-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDTGo can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDTGo reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.
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