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  • 1.
    Artursson, Elisabet
    et al.
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Andersson, Per Ola
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Akfur, Christine
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Börjegren, Susanne
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Ekström, Fredrik
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå.
    Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: Possible implications for the HI-6 antidote substrate specificity2013In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 85, no 9, p. 1389-1397Article in journal (Refereed)
    Abstract [en]

    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300 μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  • 2. Barbercheck Epler, Chelsea R.
    et al.
    Bullitt, Esther
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Bacterial adhesion pili2018In: Membrane protein complexes: structure and function / [ed] J. Robin Harris, Egbert Boekema, Springer Publishing Company, 2018, , p. 18p. 1-18Chapter in book (Refereed)
    Abstract [en]

    Escherichia coli bacterial cells produce multiple types of adhesion pili that mediate cell-cell and cell-host attachments. These pili (also called 'fimbriae') are large biopolymers that are comprised of subunits assembled via a sophisticated micro-machinery into helix-like structures that are anchored in the bacterial outer membrane. They are commonly essential for initiation of disease and thus provide a potential target for antibacterial prevention and treatment. To develop new therapeutics for disease prevention and treatment we need to understand the molecular mechanisms and the direct role of adhesion pili during pathogenesis. These helix-like pilus structures possess fascinating and unique biomechanical properties that have been thoroughly investigated using high-resolution imaging techniques, force spectroscopy and fluid flow chambers. In this chapter, we first discuss the structure of pili and the micro-machinery responsible for the assembly process. Thereafter, we present methods for measurement of the biomechanics of adhesion pili, including optical tweezers. Data demonstrate unique biomechanical properties of pili that allow bacteria to sustain binding during in vivo fluid shear forces. We thereafter summarize the current biomechanical findings related to adhesion pili and show that pili biomechanical properties are niche-specific. That is, the data suggest that there is an organ-specific adaptation of pili that facilitates infection of the bacteria's target tissue. Thus, pilus biophysical properties are an important part of Escherichia coli pathogenesis, allowing bacteria to overcome hydrodynamic challenges in diverse environments.

  • 3. Carbera Rodrigues, Carlos
    et al.
    Ekström, Fredrik
    Wolf, Jana
    Seibt, Henrik
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stier, Gunter
    Grundström, Christin
    Hung, Shen-Hua
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The crystal structure of the DmpR AAA+ domain suggests a novel mode of ATP-dependent transcriptional controlManuscript (preprint) (Other academic)
  • 4.
    Cisneros, David A.
    et al.
    BIOTEC, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany..
    Friedrichs, Jens
    Taubenberger, Anna
    Franz, Clemens M
    Muller, Daniel J
    Creating ultrathin nanoscopic collagen matrices for biological and biotechnological applications2007In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 3, no 6, p. 956-963Article in journal (Refereed)
    Abstract [en]

    The biofunctionalization of materials creates interfaces on which proteins, cells, or tissues can fulfill native or desired tasks. Here we report how to control the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Collagen fibrils in these ultrathin (approximately 3 nm) matrices maintained their native structure as observed in vivo. This opens up the possibility to create programmable biofunctionalized matrices using collagen-binding proteins or proteins fused with collagen-binding domains. Applied to eukaryotic cells, these nanostructured matrices can direct cellular processes such as adhesion, orientation and migration.

  • 5.
    Cisneros, David A.
    et al.
    Biotechnology Center, University of Technology Dresden, 01307 Dresden, Germany.
    Hung, Carlos
    Franz, Clemens M
    Muller, Daniel J
    Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy2006In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 154, no 3, p. 232-245Article in journal (Refereed)
    Abstract [en]

    Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.

  • 6.
    Cisneros, David A.
    et al.
    Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg 49, 01307 Dresden, Germany.
    Muller, Daniel J
    Daud, Sofian M
    Lakey, Jeremy H
    An approach to prepare membrane proteins for single-molecule imaging2006In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 45, no 20, p. 3252-3256Article in journal (Refereed)
  • 7.
    Cisneros, David A.
    et al.
    Biotechnology Center, University of Technology, Dresden, Germany.
    Oberbarnscheidt, Leoni
    Pannier, Angela
    Klare, Johann P.
    Helenius, Jonne
    Engelhard, Martin
    Oesterhelt, Filipp
    Muller, Daniel J.
    Transducer binding establishes localized interactions to tune sensory rhodopsin II2008In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 16, no 8, p. 1206-1213Article in journal (Refereed)
    Abstract [en]

    In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.

  • 8.
    Cisneros, David A.
    et al.
    BIOTEC, University of Technology, 01307 Dresden, Germany..
    Oesterhelt, Dieter
    Müller, Daniel J
    Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin2005In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 13, no 2, p. 235-242Article in journal (Refereed)
    Abstract [en]

    Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.

  • 9. Dalhus, Bjørn
    et al.
    Saarinen, Markuu
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Eklund, Pär
    Johansson, Kenth
    Karlsson, Andreas
    Ramaswamy, S
    Bjørk, Alexandra
    Synstad, Bjørnar
    Naterstad, Kristine
    Sirevåg, Reidun
    Eklund, Hans
    Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.2002In: J Mol Biol, ISSN 0022-2836, Vol. 318, no 3, p. 707-21Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.

  • 10. Dao, E. Han
    et al.
    Poitevin, Frederic
    Sierra, Raymond G.
    Gati, Cornelius
    Rao, Yashas
    Ciftci, Halil Ibrahim
    Aksit, Fulya
    McGurk, Alex
    Obrinski, Trevor
    Mgbam, Paul
    Hayes, Brandon
    de Lichtenberg, Casper
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pardo-Avila, Fatima
    Corsepius, Nicholas
    Zhang, Lindsey
    Seaberg, Matthew H.
    Hunter, Mark S.
    Liang, Mengling
    Koglin, Jason E.
    Wakatsuki, Soichi
    Demirci, Hasan
    Structure of the 30S ribosomal decoding complex at ambient temperature2018In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 24, no 12, p. 1667-1676Article in journal (Refereed)
    Abstract [en]

    The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Therm us thermophilus 30S ribosomal subunit decoding complex to 3.45 angstrom resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.

  • 11. Dongre, Mitesh
    et al.
    Singh, Naorem Santa
    Dureja, Chetna
    Peddada, Nagesh
    Solanki, Ashish K
    Ashish, Ganguly
    Raychaudhuri, Saumya
    Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: lessons from a natural variant.2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17, p. 15043-9Article in journal (Refereed)
    Abstract [en]

    HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR(V2)) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR(V2) to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR(V2) (HapR(V2G)), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR(V2) and HapR(V2G) proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

  • 12. 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.

  • 13.
    Forsgren, Nina
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Structural studies of the surface adhesin SspB from Streptococcus gordonii2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface proteins on microorganisms that build up the oral biofilm are key players in the formation of the biofilm. Antigen I/II proteins are surface adhesins found on virtually all oral streptococci and share a conserved multi-domain architecture. These adhesins bind surface components on other bacteria and on host cells. Thus, they are crucial for the development of the biofilm.    

    The objective of this thesis work is the structural characterization of the large multi-domain Antigen I/II protein SspB from the primary colonizing commensal bacterium Streptococcus gordonii.

    The crystal structure of the variable domain of SspB was determined to 2.3 Å resolution. The domain comprises a β-supersandwich and a putative binding cleft stabilized by a calcium ion. Despite high similarity in the overall structure, the cleft within SspB is significantly smaller than the cleft within the homologous protein from Streptococcus mutans, indicating that different substrates may bind in the clefts. A screen for carbohydrate binding resulted in no hits for interaction with the SspB variable domain suggesting that the cleft may not be suitable for binding sugars.

    This thesis also presents the high resolution 1.5 Å structure of a truncated C-terminal domain of SspB, the first of an Antigen I/II C-domain. The structure contains two structurally related domains, each containing one calcium ion and one intramolecular isopeptide bond. The SspB protein shares the feature of intramoleular isopeptide bonds with other surface proteins from Gram positive bacteria, such as pili from Streptococcus pyogenes and Corynebacterium diphtheriae. Intramolecular isopeptide bonds are suggested to be a common feature for retaining stability in a harsh environment. The SspB adherence region, shown to be the recognition motif for Porphyromonas gingivalis attachment to S. gordonii, protrudes from the core protein as a handle available for recognition.

    In conclusion, this thesis work has provided new knowledge about the SspB protein and increased the understanding of the common structure of AgI/II proteins.

  • 14.
    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.

  • 15.
    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.

  • 16.
    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.

  • 17.
    Huang, Shenghua
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Forsman, Cecilia
    Orphan Biovitrum AB, Umeå, Sweden.
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa: Inhibitor complexes with Anions and Acetazolamide2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12, p. e28458-Article in journal (Refereed)
    Abstract [en]

    The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

  • 18. Janovjak, Harald
    et al.
    Kedrov, Alexej
    Cisneros, David A.
    Center of Biotechnology, University of Technology and Max-Planck-Institute of Molecular Cell Biology and Genetics, Tatzberg 49, D-01307 Dresden, Germany.
    Sapra, K Tanuj
    Struckmeier, Jens
    Muller, Daniel J
    Imaging and detecting molecular interactions of single transmembrane proteins2006In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 27, no 4, p. 546-561Article in journal (Refereed)
    Abstract [en]

    Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.

  • 19.
    Karlsson, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Structural and functional properties of transthyretin2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information.

    The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.

    The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly.

    We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals.

  • 20.
    Karlsson, Göran
    et al.
    Swedish NMR Centre at the University of Gothenburg.
    Persson, Cecilia
    Swedish NMR Centre at the University of Gothenburg.
    Mayzel, Maxim
    Swedish NMR Centre at the University of Gothenburg.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Backman, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin22016In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 84, no 4, p. 461-466Article in journal (Refereed)
    Abstract [en]

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final stru-ture ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker.

  • 21.
    Lenman, Annasara
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Liaci, A. Manuel
    Liu, Yan
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Frank, Martin
    Blaum, Bärbel S.
    Chai, Wengang
    Podgorski, Iva I.
    Harrach, Balázs
    Benko, Mária
    Feizi, Ten
    Stehle, Thilo
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Polysialic acid is a cellular receptor for human adenovirus 522018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. E4264-E4273Article in journal (Refereed)
    Abstract [en]

    Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for protein-carbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.

  • 22.
    Liljas, Anders
    et al.
    Lund University (Sweden).
    Liljas, Lars
    Uppsala University (Sweden).
    Ash, Miriam-Rose
    The University of Sydney (Australia).
    Lindblom, Göran
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nissen, Paul
    Aarhus University (Denmark).
    Kjeldgaard, Morten
    Aarhus University (Denmark).
    Textbook of structural biology2017 (ed. 2)Book (Refereed)
    Abstract [en]

    This book provides a comprehensive coverage of the basic principles of structural biology, as well as an up-to-date summary of some main directions of research in the field. The relationship between structure and function is described in detail for soluble proteins, membrane proteins, membranes, and nucleic acids. There are several books covering protein structure and function, but none that give a complete picture, including nucleic acids, lipids, membranes and carbohydrates, all being of central importance in structural biology. The book covers state-of-the-art research in various areas. It is unique for its breadth of coverage by experts in the fields. The book is richly illustrated with more than 400 color figures to highlight the wide range of structures.

  • 23. Liljas, Anders
    et al.
    Liljas, Lars
    Piskur, Jure
    Lindblom, Göran
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nissen, Poul
    Kjeldgaard, Morten
    Textbook of structural biology2009 (ed. 1)Book (Other academic)
  • 24.
    Locmelis, Roland
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural biology studies of thylakoid lumen proteins required for photosystem II assembly and function2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Little is known about the structures and functions of thylakoid lumen proteins. However, some of these proteins have an essential role in photosynthesis. Photosystem II (PSII) complexes are embedded in the thylakoid membrane of oxygenic photosynthetic organisms and one of the central subunits, the D1 protein, is damaged by light during the light driven water – splitting reaction and must be replaced frequently. One of the thylakoid lumen proteins that is essential for assembly and renewal of PSII complexes is the High Chlorophyll Fluorescence 136 (HCF136) protein. Another important protein for the PSII complex assembly is the Low PSII Accumulation Protein 19 (LPA19). Both proteins, HCF136 and LPA19, were shown to bind to the core subunits of the PSII complex from the lumenal side and LPA19 has been shown to explicitly interact with the soluble C-terminus of the D1 protein, one of the core PSII complex proteins. Prior to the replacement of the damaged D1 protein, the PSII complex needs to be disassembled, which is done with the help of the Maintenance of Photosystem II under High light 2 (MPH2) protein. MPH2, also called TL16, is required during the repair cycle of the PSII complex particularly under increased and fluctuating light conditions.

    In this work I have determined the three-dimensional X-ray structures of the HCF136 protein at 1.6 Å resolution and the LPA19 protein at 1.2 Å resolution and have also biochemically analyzed possible interactions of HCF136 with the C-termini of D1 protein. In addition, we have determined the NMR structure of the MPH2 protein.

    The protein structures of HCF136, LPA19, and MPH2 determined from A. thaliana provide us with a starting point for further studies to improve our understanding of their functional roles in the assembly, maintenance, disassembly and renewal of the PSII complex. The structures are revealing the molecular details that are particularly important during the design of mutations to study protein-protein interactions and the binding of co-factors.

    Furthermore, I have contributed to the characterization of AnPrx6, the 1-Cyx peroxiredoxin from Anabaena sp. 7120. Peroxiredoxins are important caretakers of reactive oxygen species and a homolog PrxQ in A.thaliana is found in the thylakoid lumen. The dimeric AnPrx6 protein revealed different active site residues conformations in each of the dimers, which is probably coupled to its enzymatic activity. Unexpectedly, the protein acted also as a chaperone and showed chaperone activity in its dimeric state, which is a novelty for Prx proteins.

  • 25.
    Locmelis, Roland
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Storm, Patrik
    Murphy, Michael
    Schröder, Wolfgang P.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Three-dimensional structure of the Photosystem II assembly factor HCF136 from Arabidopsis thalianaManuscript (preprint) (Other academic)
  • 26.
    Locmelis, Roland
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Norman, Per-Gustav
    Sauer, Uwe H.
    Photosystem II assembly factor LPA19 from Arabidopsis thaliana: Cloning, expression, purification and X-ray diffraction analysisManuscript (preprint) (Other academic)
  • 27.
    Locmelis, Roland
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Cecilia
    Schröder, Wolfgang
    Sauer, Uwe H.
    Solution structure of the Photosystem II repair factor MPH2 from Arabidopsis thaliana: Cloning, expression, purification and structure analysisManuscript (preprint) (Other academic)
  • 28.
    Locmelis, Roland
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Stier, Gunter
    Sauer, Uwe H.
    Crystal structure of the Photosystem II assembly factor LPA19 from Arabidopsis thaliana at 1.2 Å resolutionManuscript (preprint) (Other academic)
  • 29.
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Transthyretin and the transthyretin-related protein: A structural study2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transthyretin (TTR) is one of several proteins involved in amyloid disease in humans. Unknown conformational changes of the native state of TTR result in aggregation of TTR molecules into amyloid fibrils, which accumulate in extracellular tissues. This may result in different clinical symptoms, e.g. polyneuropathy or cardiomyopathy, depending on their site of accumulation.

    Our long-term goal is to identify structural changes associated with amyloid formation. For this work, structural characterization of TTR from other species than human may provide valuable information.

    The three-dimensional X-ray crystallographic structure of TTR from sea bream (Sparus aurata) was determined at 1.75 Å resolution. Human and sea bream TTR were found to be structurally very similar. However, interesting differences were present in the area at and around -strand D, which in fish forms an extended loop region. Interestingly, this area is believed to dissociate from the structure prior to amyloid formation, to allow -strands A and B to participate in polymerization.

    During evolution, TTR from different species have developed differences in preference to their natural ligands, the thyroid hormones 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4). While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution. A significantly wider entrance and narrower thyroid hormone binding channel provide a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.

    In a separate work, we identified a novel protein family with structural similarity to TTR, which we named the transthyretin-related protein (TRP) family. To attain information about this protein family, we cloned, expressed, purified and characterized TRP from Escherichia coli (EcTRP). Furthermore, we solved the structure of EcTRP to 1.65 Å resolution. As predicted, EcTRP and human TTR are structurally very similar. Interesting structural differences are found in the area corresponding to the thyroid hormone binding site in TTR, which due to its amino acid conservation within the TRP family we identified as a putative ligand-binding site in TRPs. The function of the TRP is not known, however, recent studies suggest that it might be involved in purine catabolism.

    It has been shown that partial acid denaturation of human TTR results in amyloid-fibril formation. Interestingly, we have shown that sea bream TTR also forms amyloid-like fibrils in vitro, even though it shares only 52% sequence identity to human TTR. Corresponding studies on EcTRP did not generate amyloid-like fibrils. EcTRP has 30% sequence identity to human TTR. The fact that two of the proteins form amyloid fibrils and one does not means that they can serve as a model system for the study of amyloid formation. Further studies on these three proteins are currently performed to attain more information about the mechanism of amyloid formation.

  • 30. Melo, Arthur Alves
    et al.
    Hegde, Balachandra G.
    Shah, Claudio
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Isas, J. Mario
    Kunz, Severine
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Langen, Ralf
    Daumke, Oliver
    Structural insights into the activation mechanism of dynamin-like EHD ATPases2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. 5629-5634Article in journal (Refereed)
    Abstract [en]

    Eps15 (epidermal growth factor receptor pathway substrate 15)homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

  • 31.
    Mishra, Yogesh
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Locmelis, Roland
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nam, Kwangho
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, 76019-0065, USA.
    Söderberg, Christopher A. G.
    Storm, Patrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chaurasia, Neha
    Rai, Lal Chand
    Jansson, Stefan
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Schröder, Wolfgang P.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 71202017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 17151Article in journal (Refereed)
    Abstract [en]

    Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C-P) and, if present, the resolving Cys (C-R). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 angstrom resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

  • 32.
    Nylander, Åsa
    Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
    Structural and functional studies of streptococcal surface adhesins2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The oral cavity is home to an array of microorganisms that are associated with dental plaque. Some Gram-positive bacteria are common inhabitants of the oral cavity and in order to colonize such a unique environment adhesion becomes essential and is accomplish by adhesins expressed on the bacterial surface. Adhesins can interact with host molecules or with structures on the resident oral microbial flora. Members of the antigen I/II (AgI/II) protein family are commonly found on the surface of oral streptococci and have the unique feature that their putative adhesin domain is located in the centre of the primary sequence. Crystal structures representing parts of the C-terminal domains from two AgI/II members, SpaP from Streptococcus mutans and AspA from Streptococcus pyogenes, were determined to 2.2 and 1.8 Å resolution respectively. The structures are very similar and consist of two domains with DEv-IgG folds. The proteins are stabilized by intramolecular isopeptide bonds and tightly coordinated metal ions.

    Another group of surface proteins is the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that have their putative adhesin domain in the N-terminal, presented on a stalk formed by multiples of repeated C-terminal domains. Sgo0707 from Streptococcus gordonii is an example of this group of proteins and its N-terminal domain was determined to 2.1 Å resolution. The structure consists of two domains, N1 and N2, both of which adopt β-sandwiches. In the Sgo0707 structure no isopeptide bonds or metal ions were detected. A putative binding cleft is present in the N1 domain. Functional studies revealed collagen type-1 and keratinocytes as possible binding partners.

    In order to further characterize the AgI/II protein AspA from S. pyogenes a long form of the protein, AspA-AVPC, was expressed and purified. During the purification process it was observed that the protein fragmented into two major parts. This process could be inhibited by the addition of 0.5 mM EDTA during protein purification.

    In conclusion, these studies have resulted in adding to the knowledge of protein structures and function of streptococcal surface proteins.

  • 33.
    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)
  • 34.
    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.

  • 35.
    Olofsson, Anders
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Lindhagen-Persson, Malin
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Amide solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloid-beta(1-42) form different fibrillar structures under identical conditions.2007In: Biochem J, ISSN 1470-8728, Vol. 404, no 1, p. 63-70Article in journal (Refereed)
    Abstract [en]

    AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39–43 residue long Ab (amyloid-b)-peptide. The most abundant species, Ab(1–40) and Ab(1–42), are both present within senile plaques, but Ab(1–42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Ab-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Ab(1–40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Ab(1–42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Ab(1–40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two b-strands connected with a bend. This protection pattern partly resembles the pattern found in Ab(1–42) fibrils, but the Ab(1–40) fibrils display a significantly increased protection for the N-terminal residues Phe4–His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37–Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.

  • 36. Pantoom, Supansa
    et al.
    Yang, Aimin
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany; Institute of Chemical Biology and Precision Therapy, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
    Lift and cut: Anti-host autophagy mechanism of Legionella pneumophila2017In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 13, no 8, p. 1467-1469Article in journal (Refereed)
    Abstract [en]

    RavZ, an effector protein of pathogenic Legionella pneumophila, inhibits host macroautophagy/autophagy by deconjugation of lipidated LC3 proteins from phosphatidylethanolamine (PE) on the autophagosome membrane. The mechanism for how RavZ specifically recognizes and deconjugates the lipidated LC3s is not clear. To understand the structure-function relationship of LC3-deconjugation by RavZ, we prepared semisynthetic LC3 proteins modified with different fragments of PE or 1-hexadecanol (C16). We find that RavZ activity is strictly dependent on the conjugated PE structure and RavZ extracts LC3-PE from the membrane before deconjugation. Structural and biophysical analysis of RavZ-LC3 interactions suggest that RavZ initially recognizes LC3-PE on the membrane via its N-terminal LC3-interacting region (LIR) motif. RavZ specifically targets to autophagosome membranes by interaction with phosphatidylinositol 3-phosphate (PtdIns3P) via its C-terminal domain and association with membranes via the hydrophobic α3 helix. The α3 helix is involved in extraction of the PE moiety and docking of the fatty acid chains into the lipid-binding site of RavZ, which is related in structure to that of the phospholipid transfer protein Sec14. The LIR interaction and lipid binding facilitate subsequent proteolytic cleavage of LC3-PE. The findings reveal a novel mode of host-pathogen interaction.

  • 37.
    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.

  • 38.
    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.

  • 39. Poole, Kate
    et al.
    Khairy, Khaled
    Friedrichs, Jens
    Franz, Clemens
    Cisneros, David A.
    BioTechnological Center, University of Technology Dresden, 01307 Dresden, Germany.
    Howard, Jonathon
    Mueller, Daniel
    Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 2, p. 380-386Article in journal (Refereed)
    Abstract [en]

    Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

  • 40. Preiner, Johannes
    et al.
    Janovjak, Harald
    Rankl, Christian
    Knaus, Helene
    Cisneros, David A.
    BioTec, University of Technology Dresden, Dresden, Germany.
    Kedrov, Alexej
    Kienberger, Ferry
    Muller, Daniel J
    Hinterdorfer, Peter
    Free energy of membrane protein unfolding derived from single-molecule force measurements2007In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 93, no 3, p. 930-937Article in journal (Refereed)
    Abstract [en]

    Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.

  • 41.
    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, ISSN 1664-302X, 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.

  • 42.
    Rundqvist, Louise
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thermodynamical and structural properties of proteins and their role in food allergy2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are important building blocks of all living organisms. They are composed of a defined sequence of different amino acids, and fold into a specific three-dimensional, ordered structure. The three-dimensional structure largely determines the function of the protein, but protein function always requires motion. Small movements within the protein structure govern the functional properties, and this thesis aims to better understand these discrete protein movements. The motions within the protein structure are governed by thermodynamics, which therefore is useful to predict protein interactions.

    Nuclear magnetic resonance (NMR) is a powerful tool to study proteins at atomic resolution. Therefore, NMR is the primary method used within this thesis, along with other biophysical techniques such as Fluorescence spectroscopy, Circular Dichroism spectroscopy and in silico modeling.

    In paper I, NMR in combination with molecular engineering is used to show that the folding of the catalytical subdomains of the enzyme Adenylate kinase does not affect the core of the protein, and thus takes a first step to linking folding, thermodynamic stability and catalysis.

    In paper II, the structure of the primary allergen from Brazil nut, Ber e 1, is presented along with biophysical measurements that help explain the allergenic potential of the protein.

    Paper III describes the need for a specific Brazil nut lipid fraction needed to induce an allergenic response. NMR and fluorescence spectroscopy is used to show that there is a direct interaction between Ber e 1 and one or several components in the lipid fraction.

  • 43. Shah, D S
    et al.
    Thomas, M B
    Phillips, S
    Cisneros, David A.
    Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany‡Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany‡Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany.
    Le Brun, A P
    Holt, S A
    Lakey, J H
    Self-assembling layers created by membrane proteins on gold2007In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, no 3, p. 522-526Article in journal (Refereed)
    Abstract [en]

    Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

  • 44. Spaulding, Caitlin N.
    et al.
    Schreiber, Henry Louis
    Zheng, Weili
    Dodson, Karen W.
    Hazen, Jennie E.
    Conover, Matt S.
    Wang, Fengbin
    Svenmarker, Pontus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Luna-Rico, Areli
    Francetic, Olivera
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Hultgren, Scott
    Egelman, Edward H.
    Functional role of the type 1 pilus rod structure in mediating host-pathogen interactions2018In: eLIFE, E-ISSN 2050-084X, Vol. 7, article id e31662Article in journal (Refereed)
    Abstract [en]

    Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases.

  • 45. Taubenberger, Anna
    et al.
    Cisneros, David A.
    BioTechnological Center, University of Technology Dresden, 01307 Dresden, Germany.
    Friedrichs, Jens
    Puech, Pierre-Henri
    Muller, Daniel J
    Franz, Clemens M.
    Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy2007In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 18, no 5, p. 1634-1644Article in journal (Refereed)
    Abstract [en]

    We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.

  • 46. Tellez, Luis A.
    et al.
    Blancas-Mejia, Luis M.
    Carrillo-Nava, Ernesto
    Mendoza-Hernández, Guillermo
    Cisneros, David A.
    Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-159, 04510 México.
    Fernández-Velasco, D. Alejandro
    Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 44, p. 11665-11673Article in journal (Refereed)
    Abstract [en]

    In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.

  • 47.
    Vogl, Thomas
    et al.
    Institute of Immunology, University of Muenster, Germany.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pro-Inflammatory S100A8 and S100A9 Proteins: Self-Assembly into Multifunctional Native and Amyloid Complexes2012In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 13, no 3, p. 2893-2917Article in journal (Refereed)
    Abstract [en]

    S100A8 and S100A9 are EF-hand Ca2+ binding proteins belonging to the S100 family. They are abundant in cytosol of phagocytes and play critical roles in numerous cellular processes such as motility and danger signaling by interacting and modulating the activity of target proteins. S100A8 and S100A9 expression levels increased in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases and they are implicated in the numerous disease pathologies. The Ca2+ and Zn2+-binding properties of S100A8/A9 have a pivotal influence on their conformation and oligomerization state, including self-assembly into homo- and heterodimers, tetramers and larger oligomers. Here we review how the unique chemical and conformational properties of individual proteins and their structural plasticity at the quaternary level account for S100A8/A9 functional diversity. Additional functional diversification occurs via non-covalent assembly into oligomeric and fibrillar amyloid complexes discovered in the aging prostate and reproduced in vitro. This process is also regulated by Ca2+ and Zn2+-binding and effectively competes with the formation of the native complexes. High intrinsic amyloid-forming capacity of S100A8/A9 proteins may lead to their amyloid depositions in numerous ailments characterized by their elevated expression patterns and have additional pathological significance requiring further thorough investigation.

  • 48.
    Wang, Lixiao
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    OnD-CRF: predicting order and disorder in proteins using [corrected] conditional random fields2008In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 11, p. 1401-1402Article in journal (Refereed)
    Abstract [en]

    MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/

  • 49. Weili, Zheng
    et al.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Mortezaei, Narges
    Bullitt, Esther
    Egelman, Edward
    Cryo-EM structure of the CFA/I pilus rod2019In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 6, no 5Article in journal (Refereed)
    Abstract [en]

    Enterotoxigenic Escherichia coli (ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of class 5 pili. The helical quaternary structure of CFA/I can be unwound under tensile force and it has been shown that this mechanical property helps bacteria to withstand shear forces from fluid motion. We report in this work the CFA/I pilus structure at 4.3 Å resolution from electron cryomicroscopy (cryo-EM) data, and report details of the donor strand complementation. The CfaB pilins modeled into the cryo-EM map allow us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and chaperone–usher pili. In addition, from the model built using the EM structure we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament's structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and a point-mutated CFA/I with a propensity for unwinding. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target adhesion pili needed for initial attachment and sustained adhesion of ETEC.

  • 50.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Novel "order-disorder-order" mechanism for adenylate kinase conformational change2011Conference paper (Other academic)
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