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  • 1. Addario, Barbara
    et al.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Backman, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Characterisation of Schizosaccharomyces pombe alpha-actinin2016In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 4, article id e1858Article in journal (Refereed)
    Abstract [en]

    The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin.

  • 2.
    Backman, Lars
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The no-nonsens SDS-PAGE2018In: Schizosaccharomyces pombe: methods and protocols / [ed] Teresa L. Singleton, Humana Press, 2018, p. 89-94Chapter in book (Other academic)
    Abstract [en]

    The discontinuous polyacrylamide gel electrophoresis system devised by Laemmli (Nature 227:680–685, 1970) has not only been used in numerous laboratories but has also been modified in several ways since its birth. In our laboratories, we use a modified Laemmli SDS-PAGE system for following protein purification as well as for analysis of certain protein-protein interactions, mainly involving filametous actin.

  • 3.
    Brady, L. Jeannine
    et al.
    Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA.
    Maddocks, Sarah E.
    School of Oral and Dental Sciences, University of Bristol, Bristol BS1 2LY, UK.
    Larson, Matthew R.
    Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
    Forsgren, Nina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology.
    Deivanayagam, Champion C.
    Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
    Jenkinson, Howard F.
    School of Oral and Dental Sciences, University of Bristol, Bristol BS1 2LY, UK.
    The changing faces of Streptococcus antigen I/II polypeptide family adhesins2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 2, p. 276-286Article in journal (Refereed)
    Abstract [en]

    Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal α-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by α-helical and proline-rich regions.

    Ever since dental caries (tooth decay) was first shown to be caused by bacteria, there has been continued interest in developing vaccine or passive immunization protocols for its control or prevention (Lehner et al., 1980). Although dental caries is not fatal, and in developed countries caries is now considered to be largely avoidable through controlled diet and good oral hygiene, there remain significant problems with childhood disease, especially among indigent populations. Consequently, caries is one of the most common worldwide infectious diseases. Therefore, research continues towards employing vaccine formulations comprised of peptide components derived from surface proteins of Streptococcus mutans, a major agent associated with dental caries (Lehner et al., 1975). One of the most promising strategies seems to be delivery of peptides, derived from glucan-binding protein B (GbpB) and antigen I/II (AgI/II) protein, via a mucosal (nasal) route. The GbpB polypeptide binds extracellular glucans, thus promoting co-adhesion of S. mutans cells in the development of dental plaque (Taubman and Nash, 2006). The AgI/II protein (also named P1, SpaP, AgB or PAc) is a major surface protein that functions as an adhesin, attaching S. mutans to the saliva-coated tooth enamel surface (Koga et al., 1990; Kelly et al., 1995). Antibodies against SpaP and GbpB block adherence and co-adhesion, respectively, thus disrupting colonization of the oral cavity by S. mutans (Ma et al., 1990; 1998; Taubman and Nash, 2006).

    The terminology AgI/II derives from the identification of two major cell wall antigens I and II in S. mutans by Russell et al. (1980), and the subsequent recognition that AgII was a component of AgI. Following the discovery of AgI/II, it became apparent that genes encoding orthologous proteins were widely dispersed among the streptococci (Jenkinson and Demuth, 1997). The viridans Streptococcus AgI/II adhesins range in composition from 1310 to 1653 amino acid (aa) residues, while the Streptococcus agalactiae AgI/II proteins are smaller (826–932 aa residues) (Tettelin et al., 2005). The widespread distribution of these AgI/II protein genes across the streptococci is perhaps not surprising, given the complex streptococcal communities that exist on surfaces of the oro- and naso-pharynx and within the bacterial soup of saliva. It is interesting, though, that the AgI/II family polypeptide genes have not yet been discovered in Streptococcus pneumoniae, which might be by the fact that S. pneumoniae forms a distinct evolutionary cluster (Kilian et al., 2008).

  • 4.
    Dongre, Mitesh
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Singh, Bhupender
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Aung, Kyaw Min
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Larsson, Per
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Miftakhova, Regina R.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Askarian, Fatemeh
    Johannessen, Mona
    von Hofsten, Jonas
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Erhardt, Marc
    Tuck, Simon
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Flagella-mediated secretion of a novel Vibrio cholerae cytotoxin affecting both vertebrate and invertebrate hosts2018In: Communications Biology, ISSN 2399-3642, Vol. 1, article id 59Article in journal (Refereed)
    Abstract [en]

    Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.

  • 5.
    Drobni, Mirva
    et al.
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Hallberg, K
    Öhman, Ulla
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Birve, Anna
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Persson, Karina
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Johansson, Ingegerd
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities.2006In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 43, no 6Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. RESULTS: Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. CONCLUSION: The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

  • 6. Eriksson, Hanna M
    et al.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Zhang, Shuguang
    Wieslander, Åke
    High-yield expression and purification of a monotopic membrane glycosyltransferase2009In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 2, p. 143-148Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-dodecyl-beta-D-maltoside (DDM) (approximately 1x critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.

  • 7.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sheng, Nongfei
    Umeå University, Faculty of Medicine, Department of Odontology.
    Mårell, Lena
    Umeå University, Faculty of Medicine, Department of Odontology.
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Streptococcus Mutans Adhesin Biotypes that Match and Predict Individual Caries Development2017In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 24, p. 205-215Article in journal (Refereed)
    Abstract [en]

    Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

  • 8.
    Forsgren, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Lamont, Richard J
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB2009In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 9, p. 1896-1905Article in journal (Refereed)
    Abstract [en]

    The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

  • 9.
    Forsgren, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Lamont, Richard J
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB c-terminal domain2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 3, p. 740-751Article in journal (Refereed)
    Abstract [en]

    Streptococcus gordonii is a primary colonizer and is involved in the formation of dental plaque. This bacterium expresses several surface proteins. One of them is the adhesin SspB, which is a member of the Antigen I/II family of proteins. SspB is a large multi-domain protein that has interactions with surface molecules on other bacteria and on host cells, and is thus a key factor in the formation of biofilms. Here, we report the crystal structure of a truncated form of the SspB C-terminal domain, solved by single-wavelength anomalous dispersion to 1.5Å resolution. The structure represents the first of a C-terminal domain from a streptococcal Antigen I/II protein and is comprised of two structurally related β-sandwich domains, C2 and C3, both with a Ca2+ bound in equivalent positions. In each of the domains, a covalent isopeptide bond is observed between a lysine and an asparagine, a feature that is believed to be a common stabilization mechanism in Gram-positive surface proteins. S. gordonii biofilms contain attachment sites for the periodontal pathogen Porphyromonas gingivalis and the SspB C-terminal domain has been shown to have one such recognition motif, the SspB adherence region. The motif protrudes from the protein, and serves as a handle for attachment. The structure suggests several additional putative binding surfaces, and other binding clefts may be created when the fulllength protein is folded.

  • 10.
    Forsgren, Nina
    et al.
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Lamont, Richard
    Persson, Karina
    Umeå University, Faculty of Medicine, Odontology.
    A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis2009In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, no 7, p. 712-714Article in journal (Refereed)
    Abstract [en]

    SspB is a 1500-residue adhesin expressed on the surface of the oral bacterium Streptococcus gordonii. Its interaction with other bacteria and host cells initiates the development of dental plaque. The full-length C-terminal domain of SspB was cloned, overexpressed in Escherichia coli and purified. However, the protein could not be crystallized. Limited proteolysis of the full-length C-domain identified a core fragment. The proteolysis product was cloned, expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method. X-ray data were collected and processed to a maximum resolution of 2.1 A with 96.4% completeness. The crystals belonged to space group P2(1), with one molecule in the asymmetric unit, a solvent content of 33.7% and a corresponding Matthews coefficient of 1.85 A(3) Da(-1).

  • 11.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hasegawa, Yoshiaki
    Yoshimura, Fuminobu
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural and functional characterization of shaft, anchor, and tip proteins of the Mfa1 fimbria from the periodontal pathogen Porphyromonas gingivalis2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1793Article in journal (Refereed)
    Abstract [en]

    Very little is known about how fimbriae of Bacteroidetes bacteria are assembled. To shed more light on this process, we solved the crystal structures of the shaft protein Mfa1, the regulatory protein Mfa2, and the tip protein Mfa3 from the periodontal pathogen Porphyromonas gingivalis. Together these build up part of the Mfa1 fimbria and represent three of the five proteins, Mfa1-5, encoded by the mfa1 gene cluster. Mfa1, Mfa2 and Mfa3 have the same overall fold i.e., two β-sandwich domains. Upon polymerization, the first β-strand of the shaft or tip protein is removed by indigenous proteases. Although the resulting void is expected to be filled by a donor-strand from another fimbrial protein, the mechanism by which it does so is still not established. In contrast, the first β-strand in Mfa2, the anchoring protein, is firmly attached by a disulphide bond and is not cleaved. Based on the structural information, we created multiple mutations in P. gingivalis and analysed their effect on fimbrial polymerization and assembly in vivo. Collectively, these data suggest an important role for the C-terminal tail of Mfa1, but not of Mfa3, affecting both polymerization and maturation of downstream fimbrial proteins.

  • 12.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nylander, Åsa
    Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
    Jenkinson, H.
    University of Bristol.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes2014In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 4, p. 283-289Article in journal (Refereed)
    Abstract [en]

    The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.

  • 13.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803: novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 14.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tam, Lam Xuan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803 – Novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Abstract Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 15. Hasegawa, Y.
    et al.
    Iijima, Y.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nagano, K.
    Yoshida, Y.
    Lamont, RJ.
    Kikuchi, T.
    Mitani, A.
    Yoshimura, F.
    Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis2016In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 95, no 11, p. 1291-1297Article in journal (Refereed)
    Abstract [en]

    Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

  • 16.
    Kloppsteck, Patrik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hasegawa, Yoshiaki
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 22945Article in journal (Refereed)
    Abstract [en]

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae.

  • 17.
    Landström, Jens
    et al.
    Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rademacher, Christoph
    Institute of Chemistry, University of Luebeck.
    Lundborg, Magnus
    Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
    Wakarchuk, Warren
    National Research Council of Canada.
    Thomas, Peters
    Institute of Chemistry, University of Luebeck.
    Widmalm, Göran
    Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
    Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase2012In: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 29, no 7, p. 491-502Article in journal (Refereed)
    Abstract [en]

    The α-1,3-glucosyltransferase WaaG is involved in the synthesis of the core region of lipopolysaccharides in E. coli. A fragment-based screening for inhibitors of the WaaG glycosyltrasferase donor site has been performed using NMR spectroscopy. Docking simulations were performed for three of the compounds of the fragment library that had shown binding activity towards WaaG and yielded 3D models for the respective complexes. The three ligands share a hetero-bicyclic ring system as a common structural motif and they compete with UDP-Glc for binding. Interestingly, one of the compounds promoted binding of uridine to WaaG, as seen from STD NMR titrations, suggesting a different binding mode for this ligand. We propose these compounds as scaffolds for the design of selective high-affinity inhibitors of WaaG. Binding of natural substrates, enzymatic activity and donor substrate selectivity were also investigated by NMR spectroscopy. Molecular dynamics simulations of WaaG were carried out with and without bound UDP and revealed structural changes compared to the crystal structure and also variations in flexibility for some amino acid residues between the two WaaG systems studied.

  • 18.
    Nylander, Åsa
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Forsgren, Nina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology.
    Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans2011In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 67, p. 23-26Article in journal (Refereed)
    Abstract [en]

    SpaP is a 1500-residue adhesin expressed on the surface of the caries-implicated bacterium Streptococcus mutans. SpaP is a member of the antigen I/II (AgI/II) family of proteins expressed by oral streptococci. These surface proteins are crucial for the incorporation of streptococci into dental plaque. The structure of the C-terminal domain of SpaP (residues 1136-1489) was solved and refined to 2.2 Å resolution with six molecules in the asymmetric unit. Similar to a related AgI/II structure, SpaP is stabilized by isopeptide bonds between lysine and asparagine side chains.

  • 19.
    Nylander, Åsa
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jenkinson, H
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Expression and purification of Streptococcus pyogenes adhesin AspAManuscript (preprint) (Other academic)
  • 20.
    Nylander, Åsa
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Svensäter, Gunnel
    Malmö Högskola.
    Senadheera, Dilani B.
    University of Toronto.
    Cvitkovitch, Dennis G.
    University of Toronto.
    Davies, Julia R.
    Malmö Högskola.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural and functional analysis of the N-terminal domain of the Streptococcus gordonii adhesin Sgo07072013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e63768-Article in journal (Refereed)
    Abstract [en]

    The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

  • 21.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling2011In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. F67, p. 1207-1210Article in journal (Refereed)
    Abstract [en]

    Actinomyces oris is an oral bacterium important for the development of dental plaque. It expresses two forms of fimbriae: type 1 and type 2. FimP, which is the fimbrial protein that is polymerized into the stalk of the type 1 fimbriae, was cloned, overexpressed and crystallized. X-ray data were collected and processed to 2.2 Å resolution. The crystals belonged to space group P21212, with one molecule in the asymmetric unit. To facilitate structure determination using single anomalous dispersion, three methionines were introduced by site-directed mutagenesis. Crystals of selenomethionine-labelled protein were obtained by streak-seeding and diffracted to 2.0 Å resolution.

  • 22.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology.
    Structure of the sortase AcSrtC-1 from Actinomyces oris2011In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, p. 212-217Article in journal (Refereed)
    Abstract [en]

    The crystal structure of the sortase AcSrtC-1 from the oral microorganism Actinomyces oris has been determined to 2.4 Å resolution. AcSrtC-1 is a cysteine transpeptidase that is responsible for the formation of fimbriae by the polymerization of a shaft protein. Similar to other pili-associated sortases, the AcSrtC-1 active site is protected by a flexible lid. The asymmetric unit contains five AcSrtC-1 molecules and their catalytic Cys-His-Arg triads are trapped in two different conformations. It is also shown that the thermostability of the enzyme is increased by the presence of calcium.

  • 23.
    Persson, Karina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Backman, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Crystallization of recombinant α-actinin and related proteins2018In: Schizosaccharomyces pombe: methods and protocols / [ed] Teresa L. Singleton, Humana Press, 2018, p. 95-103Chapter in book (Other academic)
    Abstract [en]

    When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is possible to crystallize. Still, by following some simple checkpoints the chances of obtaining crystals are increased. The primary checkpoints are purity, stability, concentration, and homogeneity. High-quality protein crystals are needed. This protocol will allow an investigator to: clone, express, and crystallize a protein of interest.

  • 24.
    Persson, Karina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    The pilin protein FimP from Actinomyces oris: crystal structure and sequence analyses2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, p. e48364-Article in journal (Refereed)
    Abstract [en]

    The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.

  • 25.
    Persson, Karina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Heidler, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hasegawa, Yoshiaki
    Structural studies of the five pilin proteins building up the type-V pilus Mfa1 of Porphyromonas gingivalis2018In: Acta Crystallographica - Section A : Foundations and Advances, ISSN 2053-2733, Vol. 74, p. E425-E425Article in journal (Other academic)
  • 26. Ye, L
    et al.
    Li, YL
    Mellström, K
    Mellin, C
    Bladh, LG
    Koehler, K
    Garg, N
    Garcia Collazo, AM
    Litten, C
    Husman, B
    Persson, Karina
    Umeå University, Faculty of Medicine, Odontology.
    Ljunggren, J
    Grover, G
    Sleph, PG
    George, R
    Malm, J
    Thyroid receptor ligands. 1. Agonist ligands selective for the thyroid receptor beta1.2003In: Journal of Medicinal Chemistry, Vol. 24;46, no 9, p. 1580-8Article in journal (Refereed)
    Abstract [en]

    Endogenous thyroid receptor hormones 3,5,3',5'-tetraiodo-l-thyronine (T(4), 1) and 3,5,3'-triiodo-l-thyronine (T(3), 2) exert a significant effects on growth, development, and homeostasis in mammals. They regulate important genes in intestinal, skeletal, and cardiac muscles, the liver, and the central nervous system, influence overall metabolic rate, cholesterol and triglyceride levels, and heart rate, and affect mood and overall sense of well being. The literature suggests many or most effects of thyroid hormones on the heart, in particular on the heart rate and rhythm, are mediated through the TRalpha(1) isoform, while most actions of the hormones on the liver and other tissues are mediated more through the TRbeta(1) isoform of the receptor. Some effects of thyroid hormones may be therapeutically useful in nonthyroid disorders if adverse effects can be minimized or eliminated. These potentially useful features include weight reduction for the treatment of obesity, cholesterol lowering for treating hyperlipidemia, amelioration of depression, and stimulation of bone formation in osteoporosis. Prior attempts to utilize thyroid hormones pharmacologically to treat these disorders have been limited by manifestations of hyperthyroidism and, in particular, cardiovascular toxicity. Consequently, development of thyroid hormone receptor agonists that are selective for the beta-isoform could lead to safe therapies for these common disorders while avoiding cardiotoxicity. We describe here the synthesis and evaluation of a series of novel TR ligands, which are selective for TRbeta(1) over TRalpha(1). These ligands could potentially be useful for treatment of various disorders as outlined above. From a series of homologous R(1)-substituted carboxylic acid derivatives, increasing chain length was found to have a profound effect on affinity and selectivity in a radioreceptor binding assay for the human thyroid hormone receptors alpha(1) and beta(1) (TRalpha(1) and TRbeta(2)) as well as a reporter cell assay employing CHOK1-cells (Chinese hamster ovary cells) stably transfected with hTRalpha(1) or hTRbeta(1) and an alkaline phosphatase reporter-gene downstream thyroid response element (TRAFalpha(1) and TRAFbeta(1)). Affinity increases in the order formic, acetic, and propionic acid, while beta-selectivity is highest when the R(1) position is substituted with acetic acid. Within this series 3,5-dibromo-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (11a) and 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (15) were found to reveal the most promising in vitro data based on isoform selectivity and were selected for further in vivo studies. The effect of 2, 11a, and 15 in a cholesterol-fed rat model was monitored including potencies for heart rate (ED(15)), cholesterol (ED(50)), and TSH (ED(50)). Potency for tachycardia was significantly reduced for the TRbeta selective compounds 11a and 15 compared with 2, while both 11a and 15 retained the cholesterol-lowering potency of 2. This left an approximately 10-fold therapeutic window between heart rate and cholesterol, which is consistent with the action of ligands that are approximately 10-fold more selective for TRbeta(1). We also report the X-ray crystallographic structures of the ligand binding domains of TRalpha and TRbeta in complex with 15. These structures reveal that the single amino acid difference in the ligand binding pocket (Ser277 in TRalpha or Asn331 in TRbeta) results in a slightly different hydrogen bonding pattern that may explain the increased beta-selectivity of 15.

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