SwePub - search: WFRF:(Berntsson Ronnie)

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1.	Berntsson, Ronnie P. -A., et al. (author) Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2 2014 In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2725-2735 Journal article (peer-reviewed)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.
2.	Berntsson, Ronnie P. A., et al. (author) Structure of dual receptor binding to botulinum neurotoxin B 2013 In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4, s. 2058- Journal article (peer-reviewed)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.
3.	Berntsson, Ronnie P., et al. (author) Crystal Structures of Botulinum Neurotoxin DC in Complex with Its Protein Receptors Synaptotagmin I and II 2013 In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21:9, s. 1602-1611 Journal article (peer-reviewed)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.
4.	Breidenstein, Annika, et al. (author) PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs 2024 In: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 7:8 Journal article (peer-reviewed)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.
5.	Breidenstein, Annika, et al. (author) Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing 2023 In: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:4 Journal article (peer-reviewed)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.
6.	Carreras-Puigvert, Jordi, et al. (author) A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family 2017 In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1 Journal article (peer-reviewed)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.
7.	Dunny, Gary, et al. (author) Enterococcal sex pheromones : evolutionary pathways to complex, two-signal systems 2016 In: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 198:11, s. 1556-1562 Research review (peer-reviewed)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.
8.	Ehrenbolger, Kai Patric, 1991- (author) Nanoscopic adventures : unraveling macromolecular complexes in infectious diseases via integrative structural biology 2024 Doctoral thesis (other academic/artistic)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.
9.	Espaillat, Akbar, master, 1988-, et al. (author) A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria 2024 In: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1 Journal article (peer-reviewed)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.
10.	Frykholm, Karolin, 1977, et al. (author) DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels 2016 In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:15, s. 7219-7227 Journal article (peer-reviewed)abstract The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and W Phi affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and W Phi Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.
11.	Gad, Helge, et al. (author) MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool 2014 In: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221 Journal article (peer-reviewed)abstract Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
12.	Gourdon, Pontus Emanuel, 1978, et al. (author) Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump 2008 In: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 58:1, s. 103-113 Journal article (peer-reviewed)abstract Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0 mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5 mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.
13.	Gustafsson, Robert, et al. (author) Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster 2017 In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 591:22, s. 3781-3792 Journal article (peer-reviewed)abstract 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.
14.	Hamark, Christoffer, et al. (author) Glycans Confer Specificity to the Recognition of Ganglioside Receptors by Botulinum Neurotoxin A 2017 In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 139:1, s. 218-230 Journal article (peer-reviewed)abstract The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD la can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.
15.	Hamark, Christoffer, 1983-, et al. (author) Recognition of Ganglioside Receptors by Botulinum Neurotoxin A Other publication (other academic/artistic)
16.	Irazoki, Oihane, et al. (author) D-amino acids signal a stress-dependent run-away response in Vibrio cholerae 2023 In: Nature Microbiology. - : Springer Nature. - 2058-5276. ; 8:8, s. 1549-1560 Journal article (peer-reviewed)abstract To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.
17.	Jäger, Franziska, et al. (author) Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family 2022 In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:6, s. 876-885.e5 Journal article (peer-reviewed)abstract Multidrug-resistant bacteria pose serious problems in hospital-acquired infections (HAIs). Most antibiotic resistance genes are acquired via conjugative gene transfer, mediated by type 4 secretion systems (T4SS). Although most multidrug-resistant bacteria responsible for HAIs are of Gram-positive origin, with enterococci being major contributors, mostly Gram-negative T4SSs have been characterized. Here, we describe the structure and organization of PrgL, a core protein of the T4SS channel, encoded by the pCF10 plasmid from Enterococcus faecalis. The structure of PrgL displays similarity to VirB8 proteins of Gram-negative T4SSs. In vitro experiments show that the soluble domain alone is enough to drive both dimerization and dodecamerization, with a dimerization interface that differs from all other known VirB8-like proteins. In vivo experiments verify the importance of PrgL dimerization. Our findings provide insight into the molecular building blocks of Gram-positive T4SS, highlighting similarities but also unique features in PrgL compared to other VirB8-like proteins.
18.	Järvå, Michael A., et al. (author) Polymer Adhesin Domains in Gram-Positive Cell Surface Proteins 2020 In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 11 Research review (peer-reviewed)abstract Surface proteins in Gram-positive bacteria are often involved in biofilm formation, host-cell interactions, and surface attachment. Here we review a protein module found in surface proteins that are often encoded on various mobile genetic elements like conjugative plasmids. This module binds to different types of polymers like DNA, lipoteichoic acid and glucans, and is here termed polymer adhesin domain. We analyze all proteins that contain a polymer adhesin domain and classify the proteins into distinct classes based on phylogenetic and protein domain analysis. Protein function and ligand binding show class specificity, information that will be useful in determining the function of the large number of so far uncharacterized proteins containing a polymer adhesin domain.
19.	Kmiec, Beata, et al. (author) Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts 2013 In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:40, s. E3761-E3769 Journal article (peer-reviewed)abstract Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.
20.	Lassinantti, Lena, et al. (author) Enterococcal PrgU Provides Additional Regulation of Pheromone-Inducible Conjugative Plasmids 2021 In: mSphere. - : American Society for Microbiology (ASM). - 2379-5042. ; 6:3 Journal article (peer-reviewed)abstract Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its type 4 secretion system (T4SS) genes, controlled by the PQ promoter. Transcription from the PQ promoter is tightly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single-cell and population levels. PrgU function requires a specific RNA sequence within an intergenic region (IGR) about 400 bp downstream of PQ. PrgU interaction with the IGR reduces levels of downstream transcripts. Single-cell expression analysis showed that cells expressing prgU decreased transcript levels more rapidly than isogenic prgU-minus cells. PrgU bound RNA in vitro without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors for selective binding in vivo. PrgU binding to its IGR target might recruit RNase(s) for targeted degradation of downstream transcripts or reduce elongation of nascent transcripts beyond the IGR.IMPORTANCE: Bacteria utilize type 4 secretion systems (T4SS) to efficiently transfer DNA between donor and recipient cells, thereby spreading genes encoding antibiotic resistance as well as various virulence factors. Regulation of expression of the T4SS proteins and surface adhesins in Gram-positive bacteria is crucial, as some of these are highly toxic to the cell. The significance of our research lies in identifying the novel mechanism by which PrgU performs its delicate fine-tuning of the expression levels. As prgU orthologs are present in various conjugative plasmids and transposons, our results are likely relevant to understanding of diverse clinically important transfer systems.
21.	Lassinantti, Lena, 1993- (author) Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems 2022 Doctoral thesis (other academic/artistic)abstract Hospital acquired (i.e. nosocomial) infections and antibiotic resistance are large issues in the world today, with about 1.3 million people estimated to have died from antibiotic resistant infections in 2019 alone, and these problems are on the rise. Type 4 Secretion Systems (T4SSs) are complex nanomachineries commonly found on conjugative plasmids. T4SSs are a major route for the translocation of genes encoding for antibiotic resistance and other virulence factors. These systems have primarily been studied in Gram-negative (G-) bacteria even though Gram-positive (G+) bacteria stand for about half of the nosocomial infections. To develop ways to limit the spread of both antibiotic resistance and virulence factors, we need to gain fundamental knowledge of T4SSs in G+ bacteria.Our work has focused on the conjugative plasmid pCF10 from the G+ bacteria Enterococcus faecalis where all the genes needed for the T4SS are under the regulation of one promoter named PQ. Most G+ T4SSs consist of three groups of proteins, namely the DNA transfer and replication (Dtr) proteins, the channel proteins and the adhesin proteins. In my work, I have focused my attention specifically on i) the regulatory protein PrgU, ii) the Dtr protein PcfF, and iii) the adhesin protein PrgB. These three proteins provide insights into three different parts of the T4SS. PrgU is part of the regulatory process of T4SS expression and has been shown to inhibit cell-toxicity mitigated by PrgB. The Dtr protein PcfF is needed for the formation of the relaxosome complex critical for conjugative transfer of the plasmid, and PrgB is involved in cellular aggregation events and is also a known virulence factor. Interestingly, increased levels of PrgB have been shown to be toxic to the cells. To inhibit PrgB induced cell toxicity, its production needs to be tightly regulated.The aims of my PhD thesis were to examine conjugation complexes belonging to Type 4 Secretion Systems in Gram-positive bacteria and to determine their function, molecular structures, and regulation. By using a combination of in vivo and in vitro methods we have; i) showed that PrgU binds to the IGR located downstream of the PQ promoter, and that the deletion of prgU in pCF10 containing cells produces increased mRNA levels of the full prgQ transcript, ii) solved the crystal structure of PcfF and identified residues that are important for the interaction with the relaxase and the origin of transfer (oriT) DNA in vitro, and confirmed this by biochemical assays and, iii) solved the entire structure of PrgB using a combination of X-ray crystallography and cryo-EM and performed in vivo assays to confirm its functions.
22.	Martínez-Carranza, Markel, 1992-, et al. (author) Synaptotagmin Binding to Botulinum Neurotoxins 2020 In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 59:4, s. 491-498 Journal article (peer-reviewed)abstract 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.
23.	Peng, Lisheng, et al. (author) Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins 2012 In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 125:13, s. 3233-3242 Journal article (peer-reviewed)abstract Botulinum neurotoxins (BoNTs) are classified into seven types (A-G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.
24.	Rahal, Rima-Maria, et al. (author) Quality research needs good working conditions 2023 In: Nature Human Behaviour. - : Nature Publishing Group. - 2397-3374. ; 7, s. 164-167 Journal article (peer-reviewed)
25.	Rehman, Saima, et al. (author) Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence 2019 In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 10 Journal article (peer-reviewed)abstract The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homodimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.
26.	Schmitt, Andreas, et al. (author) Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation 2020 In: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 432:20, s. 5681-5695 Journal article (peer-reviewed)abstract Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.
27.	Schmitt, Andreas, et al. (author) PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction 2018 In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 109:3, s. 291-305 Journal article (peer-reviewed)abstract Gram-positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram-positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono- or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell-cell contacts through alternative binding of cell-bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra- or inter-species T4SS-mediated gene transfer.
28.	Segawa, Takaya, et al. (author) Two ABC transport systems carry out peptide uptake in Enterococcus faecalis : Their roles in growth and in uptake of sex pheromones 2021 In: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 116:2, s. 459-469 Journal article (peer-reviewed)abstract Enterococcal pheromone-inducible plasmids encode a predicted OppA-family secreted lipoprotein. In the case of plasmid pCF10, the protein is PrgZ, which enhances the mating response to cCF10 pheromone. OppA proteins generally function with associated OppBCDF ABC transporters to import peptides. In this study, we analyzed the potential interactions of PrgZ with two host-encoded Opp transporters using two pheromone-inducible fluorescent reporter constructs. Based on our results, we propose renaming these loci opp1 (OG1RF_10634-10639) and opp2 (OG1RF_12366-12370). We also examined the ability of the Opp1 and Opp2 systems to mediate import in the absence of PrgZ. Cells expressing PrgZ were able to import pheromone if either opp1 or opp2 was functional, but not if both opp loci were disrupted. In the absence of PrgZ, pheromone import was dependent on a functional opp2 system, including opp2A. Comparative structural analysis of the peptide-binding pockets of PrgZ, Opp1A, Opp2A, and the related Lactococcus lactis OppA protein, suggested that the robust pheromone-binding ability of PrgZ relates to a nearly optimal fit of the hydrophobic peptide, whereas binding ability of Opp2A likely results from a more open, promiscuous peptide-binding pocket similar to L. lactis OppA.
29.	Sun, Wei-Sheng, et al. (author) New structural insights provide mechanistic understanding of enterococcal PrgB conjugation, biofilm formation and virulence Other publication (other academic/artistic)
30.	Sun, Wei-Sheng, et al. (author) Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation, and virulence 2023 In: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12 Journal article (peer-reviewed)abstract Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating-pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation, and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin (Ig)-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall.
31.	Tao, Liang, et al. (author) Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors 2017 In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8 Journal article (peer-reviewed)abstract Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits similar to 11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.
32.	Teixeira, Pedro F., et al. (author) Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin 2018 In: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 430:3, s. 348-362 Journal article (peer-reviewed)abstract Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLNE475Q in complex with the products of neurotensin cleavage at 2.7 Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1–40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35–40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.
33.	Verma, Apoorv, et al. (author) Insights into the evolution of enzymatic specificity and catalysis : from Asgard archaea to human adenylate kinases 2022 In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:44 Journal article (peer-reviewed)abstract Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.
34.	Zhang, Sicai, et al. (author) Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC 2017 In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8 Journal article (peer-reviewed)abstract Botulinum neurotoxins (BoNTs), the most potent toxins known, are potential bioterrorism agents. It is well established that all seven serotypes of BoNTs (BoNT/A-G) require complex gangliosides as co-receptors. Here, we report that BoNT/DC, a presumed mosaic toxin between BoNT/D and BoNT/C1, binds and enters efficiently into neurons lacking complex gangliosides and shows no reduction in toxicity in mice deficient in complex gangliosides. The co-crystal structure of BoNT/DC with sialyl-Thomsen-Friedenreich antigen (Sialyl-T) suggests that BoNT/DC recognizes only the sialic acid, but not other moieties in gangliosides. Using liposome flotation assays, we demonstrate that an extended loop in BoNT/DC directly interacts with lipid membranes, and the co-occurring sialic acid binding and loop-membrane interactions mediate the recognition of gangliosides in membranes by BoNT/DC. These findings reveal a unique mechanism for cell membrane recognition and demonstrate that BoNT/DC can use a broad range of sialic acid-containing moieties as co-receptors.

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