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Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Elisabeth Sauer-Eriksson)ORCID iD: 0000-0003-4777-7629
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Bernt Eric Uhlin)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2010 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, no Pt 3, p. 337-341Article in journal (Refereed) Published
Abstract [en]

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.

Place, publisher, year, edition, pages
2010. Vol. 66, no Pt 3, p. 337-341
Keywords [en]
fimbriae, FocB, transcription factors
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-27753DOI: 10.1107/S1744309110002204ISI: 000275057700031PubMedID: 20208176Scopus ID: 2-s2.0-77749237373OAI: oai:DiVA.org:umu-27753DiVA, id: diva2:277502
Available from: 2009-11-19 Created: 2009-11-19 Last updated: 2024-07-02Bibliographically approved
In thesis
1. Molecular analysis of transcription factors in uropathogenic E. coli adhesin operons
Open this publication in new window or tab >>Molecular analysis of transcription factors in uropathogenic E. coli adhesin operons
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Molekylär analys av transkriptionsfaktorer i adhesin operon hos uropatogena E. coli
Abstract [en]

The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2009. p. 63
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1304
Keywords
UPEC, E. coli, F1C, P fimbriae, cross-talk, FocB, PapB, crystal structure, OMVs
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-27757 (URN)978-91-7264-879-1 (ISBN)
Public defence
2009-12-16, Major Groove, Institutionen för Molekylärbiologi, Byggn. 6L, Norrlands Universitetssjukhus, Umeå, 10:00 (English)
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Supervisors
Available from: 2009-11-26 Created: 2009-11-19 Last updated: 2018-01-12Bibliographically approved
2. Structural studies of FocB and Transthyretin
Open this publication in new window or tab >>Structural studies of FocB and Transthyretin
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The molecular structure of a protein decides its function, its way to interact with other molecules. Using X-ray crystallography methods, a 3-dimensional, atomic model of a macromolecule can be determined. In this thesis work, the X-ray structures of two different proteins involved in human diseases were studied: FocB, which is associated with urinary tract infections, and transthyretin, which is the causative of hereditary systemic transthyretin amyloidosis.

FocB is a 12 kDa protein which binds DNA in an oligomeric fashion. It is involved in the regulation of the expression of bacterial surface organelles (fimbriae), responsible for the adhesion to specific receptors in host tissue. Specifically, FocB regulates the expression of one fimbrial type found in uropathogenic E. coli (UPEC): F1C. Our FocB structure revealed it to be an all-alpha helical protein with an atypical helix-turn-helix (HTH) motif. Residues previously found important for DNA-binding in the FocB homologue PapB, were not located in the putative “recognition helix” of the HTH-motif. FocB was also found to bind to the minor groove of the DNA. Together with homology searches showing that the DNA-interactions possible for FocB are greatly diversified, these findings indicated a DNA-interaction different from the typical DNA-interaction of a HTH-protein.

Transthyretin (TTR) is a plasma protein involved in transport of thyroxin (T4) and retinol. Mutated TTR is also the cause of the neurodegenerative disease hereditary systemic transthyretin amyloidosis, which is characterized by systemic deposition of TTR amyloid fibrils. The amyloid occurs through a process of TTR tetramer destabilization and partial unfolding. A common way to inhibit amyloid formation is to design small molecules that bind unoccupied thyroxin binding sites and stabilize the tetrameric form of the protein. The structural characterization of the binding of chloride and iodide ions to TTR revealed that two of three previously identified halogen binding pockets in the T4-binding site were just as optimal for halide binding. In addition, a third halide-binding site, bridging two TTR subunits, was found. In biochemical experiments, chloride and iodide ions were shown to stabilize the TTR structure and inhibit the TTR aggregation and/or amyloid formation, with iodide ions doing so more efficiently than the chloride ions. In the search for new TTR amyloid-inhibiting drugs, the identified halide-binding sites in the T4-binding pocket are possible starting points for structure-based drug design.

Place, publisher, year, edition, pages
Umeå: Kemiska institutionen, Umeå universitet, 2010. p. 78
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-36208 (URN)978-91-7459-057-9 (ISBN)
Public defence
2010-10-16, KBC-huset, "Lilla Hörsalen", Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-09-24 Created: 2010-09-22 Last updated: 2024-07-02Bibliographically approved

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Wikström Hultdin, UlrikaLindberg, StinaGrundström, ChristinAllgardsson, AndersHuang, ShenghuaStier, GünterÖhman, AndersUhlin, Bernt EricSauer-Eriksson, Elisabeth

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Wikström Hultdin, UlrikaLindberg, StinaGrundström, ChristinAllgardsson, AndersHuang, ShenghuaStier, GünterÖhman, AndersUhlin, Bernt EricSauer-Eriksson, Elisabeth
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Acta Crystallographica. Section F: Structural Biology and Crystallization Communications
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