Structural studies of FocB and Transthyretin
Abstract: 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.
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