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The effect of iodide and chloride on transthyretin structure and stability
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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.

Place, publisher, year, edition, pages
2005. Vol. 44, no 26, 9290-9299 p.
Keyword [en]
Chlorides/*chemistry, Crystallization, Humans, Hydrogen-Ion Concentration, Iodides/*chemistry, Models; Molecular, Prealbumin/*chemistry/isolation & purification, Protein Conformation, Protein Denaturation, Urea/chemistry
Identifiers
URN: urn:nbn:se:umu:diva-3579DOI: 10.1021/bi050249zPubMedID: 15981995OAI: oai:DiVA.org:umu-3579DiVA: diva2:142350
Available from: 2004-02-05 Created: 2004-02-05 Last updated: 2010-09-24Bibliographically approved
In thesis
1. Transthyretin from a structural perspective
Open this publication in new window or tab >>Transthyretin from a structural perspective
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Transthyretin ur ett strukturellt perspektiv
Abstract [en]

Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation.

Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.

We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.

The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet), 2004. 50 p.
Keyword
Cell and molecular biology, X-ray crystallography, amyloidosis, structural comparison, anomalous diffraction, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-190 (URN)91-7305-593-X (ISBN)
Public defence
2004-02-27, Major Groove, 6L Sjukhusområdet, Umeå Universitet 901 87, Umeå, 10:00
Opponent
Supervisors
Available from: 2004-02-05 Created: 2004-02-05 Last updated: 2017-01-24Bibliographically 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. 78 p.
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: 2010-09-24Bibliographically approved

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Wikström Hultdin, UlrikaOlofsson, AndersSauer-Eriksson, Elisabeth

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