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  • 1.
    Breidenstein, Annika
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing2023In: Life Science Alliance, E-ISSN 2575-1077, Vol. 6, no 4, article id e202201775Article in journal (Refereed)
    Abstract [en]

    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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  • 2. Dunny, Gary
    et al.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enterococcal sex pheromones: evolutionary pathways to complex, two-signal systems2016In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, no 11, p. 1556-1562Article, review/survey (Refereed)
    Abstract [en]

    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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  • 3. Frykholm, Karolin
    et al.
    Berntsson, Ronnie Per-Arne
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    Claesson, Magnus
    de Battice, Laura
    Odegrip, Richard
    Stenmark, Pål
    Westerlund, Fredrik
    DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 15, p. 7219-7227Article in journal (Refereed)
    Abstract [en]

    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.

  • 4. Gustafsson, Robert
    et al.
    Berntsson, Ronnie P.-A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Martínez-Carranza, Markel
    El Tekle, Geniver
    Odegrip, Richard
    Johnson, Eric A.
    Stenmark, Pål
    Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 22, p. 3781-3792Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 angstrom, 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.

  • 5. Hamark, Christoffer
    et al.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
    Masuyer, Geoffrey
    Henriksson, Linda M.
    Gustafsson, Robert
    Stenmark, Pal
    Widmalm, Goran
    Glycans Confer Specificity to the Recognition of Ganglioside Receptors by Botulinum Neurotoxin A2017In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 1, p. 218-230Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Irazoki, Oihane
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mateus, André
    Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
    Colin, Remy
    Max Planck Institute for Terrestrial Microbiology, and Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
    Typas, Athanasios
    Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
    Savitski, Mikhail M.
    Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
    Sourjik, Victor
    Max Planck Institute for Terrestrial Microbiology, and Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
    Berntsson, Ronnie P.-A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    D-amino acids signal a stress-dependent run-away response in Vibrio cholerae2023In: Nature Microbiology, E-ISSN 2058-5276, Vol. 8, no 8, p. 1549-1560Article in journal (Refereed)
    Abstract [en]

    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.

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  • 7.
    Jäger, Franziska
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lamy, Anaïs
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Sun, Wei-Sheng
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Guerini, Nina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family2022In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 30, no 6, p. 876-885.e5Article in journal (Refereed)
    Abstract [en]

    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.

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  • 8.
    Järvå, Michael A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hirt, Helmut
    Dunny, Gary M.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Polymer Adhesin Domains in Gram-Positive Cell Surface Proteins2020In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 11, article id 599899Article, review/survey (Refereed)
    Abstract [en]

    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.

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  • 9.
    Lassinantti, Lena
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Camacho, Martha I.
    Department of Microbiology and Molecular Genetics, McGovern Medical School, TX, Houston, United States.
    Erickson, Rebecca J B
    Department of Microbiology and Immunology, University of Minnesota, MN, Minneapolis, United States.
    Willett, Julia L E
    Department of Microbiology and Immunology, University of Minnesota, MN, Minneapolis, United States.
    De Lay, Nicholas R.
    Department of Microbiology and Molecular Genetics, McGovern Medical School, TX, Houston, United States.
    Ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dunny, Gary M.
    Department of Microbiology and Immunology, University of Minnesota, MN, Minneapolis, United States.
    Christie, Peter J.
    Department of Microbiology and Molecular Genetics, McGovern Medical School, TX, Houston, United States.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Enterococcal PrgU Provides Additional Regulation of Pheromone-Inducible Conjugative Plasmids2021In: mSphere, E-ISSN 2379-5042, Vol. 6, no 3, article id e0026421Article in journal (Refereed)
    Abstract [en]

    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.

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  • 10. Martínez-Carranza, Markel
    et al.
    Blasco, Pilar
    Gustafsson, Robert
    Dong, Min
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Widmalm, Göran
    Stenmark, Pål
    Synaptotagmin Binding to Botulinum Neurotoxins2020In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 59, no 4, p. 491-498Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt- II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide's toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.

  • 11.
    Rahal, Rima-Maria
    et al.
    Behavioral Law & Economics, Max Planck Institute for Research on Collective Goods, Bonn, Germany.
    Fiedler, Susann
    Department Strategy & Innovation, Vienna University of Economics and Business, Vienna, Austria.
    Adetula, Adeyemi
    Department of Psychology, Alex Ekwueme Federal University Ndufu-Alike, Abakaliki, Nigeria; Laboratoire Interuniversitaire de Psychologie, Personnalité, Cognition, Changement Social, Université Grenoble Alpes, Grenoble, France.
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Dirnagl, Ulrich
    QUEST Center for Responsible Research, Berlin Institute of Health, Berlin, Germany.
    Feld, Gordon B.
    Central Institute of Mental Health, Heidelberg University, Mannheim, Germany.
    Fiebach, Christian J.
    Department of Psychology, Goethe University Frankfurt, Frankfurt am Main, Germany.
    Himi, Samsad Afrin
    Department of Psychology, Jagannath University, Dhaka, Bangladesh.
    Horner, Aidan J.
    Department of Psychology, University of York, York, United Kingdom; York Biomedical Research Institute, University of York, York, United Kingdom.
    Lonsdorf, Tina B.
    Department of Systems Neuroscience, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
    Schönbrodt, Felix
    Department of Psychology, LMU Munich, München, Germany.
    Silan, Miguel Alejandro A.
    Development, Individual, Process, Handicap, Education (DIPHE) Research Unit, Université Lumière Lyon 2, Lyon, France; Annecy Behavioral Science Lab, Menthon-Saint-Bernard, France.
    Wenzler, Michael
    Tübingen, Germany.
    Azevedo, Flávio
    Department of Psychology, University of Cambridge, Cambridge, United Kingdom.
    Quality research needs good working conditions2023In: Nature Human Behaviour, E-ISSN 2397-3374, Vol. 7, p. 164-167Article in journal (Refereed)
  • 12.
    Rehman, Saima
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Li, Yang Grace
    Schmitt, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lassinantti, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Christie, Peter J.
    Berntsson, Ronnie P. -A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence2019In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 10, article id 958Article in journal (Refereed)
    Abstract [en]

    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.

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  • 13.
    Schmitt, Andreas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hirt, Helmut
    Järvå, Michael A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sun, Wei-Sheng
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dunny, Gary M.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation2020In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 432, no 20, p. 5681-5695Article in journal (Refereed)
    Abstract [en]

    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. 

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  • 14.
    Schmitt, Andreas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jiang, Kai
    Camacho, Martha I.
    Jonna, Venkateswara Rao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westerlund, Fredrik
    Christie, Peter J.
    Berntsson, Ronnie P-A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 109, no 3, p. 291-305Article in journal (Refereed)
    Abstract [en]

    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.

  • 15.
    Segawa, Takaya
    et al.
    Department of Microbiology and Immunology, University of Minnesota Medical School, MN, Minneapolis, United States.
    Johnson, Christopher M.
    Department of Microbiology and Immunology, University of Minnesota Medical School, MN, Minneapolis, United States; Recursion, UT, Salt Lake City, United States.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Dunny, Gary M.
    Department of Microbiology and Immunology, University of Minnesota Medical School, MN, Minneapolis, United States.
    Two ABC transport systems carry out peptide uptake in Enterococcus faecalis: Their roles in growth and in uptake of sex pheromones2021In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 116, no 2, p. 459-469Article in journal (Refereed)
    Abstract [en]

    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.

  • 16.
    Sun, Wei-Sheng
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Lassinantti, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Järvå, Michael A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmitt, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    New structural insights provide mechanistic understanding of enterococcal PrgB conjugation, biofilm formation and virulenceManuscript (preprint) (Other academic)
  • 17.
    Sun, Wei-Sheng
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Lassinantti, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Järvå, Michael A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmitt, Andreas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation, and virulence2023In: eLIFE, E-ISSN 2050-084X, Vol. 12Article in journal (Refereed)
    Abstract [en]

    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.

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  • 18. Tao, Liang
    et al.
    Peng, Lisheng
    Berntsson, Ronnie P. -A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
    Liu, Sai Man
    Park, SunHyun
    Yu, Feifan
    Boone, Christopher
    Palan, Shilpa
    Beard, Matthew
    Chabrier, Pierre-Etienne
    Stenmark, Pål
    Krupp, Johannes
    Dong, Min
    Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors2017In: Nature Communications, E-ISSN 2041-1723, Vol. 8, article id 53Article in journal (Refereed)
    Abstract [en]

    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.

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  • 19. Teixeira, Pedro F.
    et al.
    Masuyer, Geoffrey
    Pinho, Catarina M.
    Branca, Rui M. M.
    Kmiec, Beata
    Wallin, Cecilia
    Wärmländer, Sebastian K. T. S.
    Berntsson, Ronnie P. -A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ankarcrona, Maria
    Gräslund, Astrid
    Lehtiö, Janne
    Stenmark, Pål
    Glaser, Elzbieta
    Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 3, p. 348-362Article in journal (Refereed)
    Abstract [en]

    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.

  • 20.
    Verma, Apoorv
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Åberg-Zingmark, Emma
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ul Mushtaq, Ameeq
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rogne, Per
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Berntsson, Ronnie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Backman, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nam, Kwangho
    Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, USA..
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Insights into the evolution of enzymatic specificity and catalysis: from Asgard archaea to human adenylate kinases2022In: Science Advances, E-ISSN 2375-2548, Vol. 8, no 44, article id eabm4089Article in journal (Refereed)
    Abstract [en]

    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.

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  • 21. Zhang, Sicai
    et al.
    Berntsson, Ronnie P. A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.
    Tepp, William H.
    Tao, Liang
    Johnson, Eric A.
    Stenmark, Pal
    Dong, Min
    Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC2017In: Nature Communications, E-ISSN 2041-1723, Vol. 8, article id 1637Article in journal (Refereed)
    Abstract [en]

    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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