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Structural studies of FocB and Transthyretin
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Grp Elisabeth Sauer-Eriksson)
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. , 78 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-36208ISBN: 978-91-7459-057-9 (print)OAI: oai:DiVA.org:umu-36208DiVA: diva2:352702
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: 2010-09-24Bibliographically approved
List of papers
1. Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
Open this publication in new window or tab >>Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
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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, 337-341 p.Article 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.

Keyword
fimbriae, FocB, transcription factors
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-27753 (URN)10.1107/S1744309110002204 (DOI)000275057700031 ()20208176 (PubMedID)
Available from: 2009-11-19 Created: 2009-11-19 Last updated: 2017-12-12Bibliographically approved
2. Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
Open this publication in new window or tab >>Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
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2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 16, 3368-3381 p.Article in journal (Refereed) Published
Abstract [en]

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form different protein-protein complexes and that they may exert both positive and negative effects on transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all alpha helical structure with a helix-turn-helix (HTH) motif. Interestingly, conserved residues important for DNA-binding are not located in the recognition helix of the HTH-motif, but in the preceding helix. Results from protein-DNA binding studies indicated that FocB interacts with minor groove of its cognate DNA, which also points to a DNA-interaction unusual for this motif. Packing interactions in the crystals gave two plausible dimerization interfaces. Conserved residues known to be important for protein oligomerization are present at both interfaces, suggesting that both sites play a role in a functional FocB protein.

Place, publisher, year, edition, pages
Wiley, 2010
Keyword
fimbriae, FocB, repressor protein, uropathogenic Escherichia coli, X-ray crystallography
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-27755 (URN)10.1111/j.1742-4658.2010.07742.x (DOI)000280631300010 ()
Available from: 2009-11-19 Created: 2009-11-19 Last updated: 2017-12-12Bibliographically approved
3. The effect of iodide and chloride on transthyretin structure and stability
Open this publication in new window or tab >>The effect of iodide and chloride on transthyretin structure and stability
2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 26, 9290-9299 p.Article in journal (Other academic) Published
Abstract [en]

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

Keyword
Chlorides/*chemistry, Crystallization, Humans, Hydrogen-Ion Concentration, Iodides/*chemistry, Models; Molecular, Prealbumin/*chemistry/isolation & purification, Protein Conformation, Protein Denaturation, Urea/chemistry
Identifiers
urn:nbn:se:umu:diva-3579 (URN)10.1021/bi050249z (DOI)15981995 (PubMedID)
Available from: 2004-02-05 Created: 2004-02-05 Last updated: 2017-12-14Bibliographically approved

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