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Transthyretin from a structural perspective
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
2004 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Transthyretin ur ett strukturellt perspektiv (Swedish)
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 [en]
Cell and molecular biology, X-ray crystallography, amyloidosis, structural comparison, anomalous diffraction
Keyword [sv]
Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-190ISBN: 91-7305-593-X (print)OAI: oai:DiVA.org:umu-190DiVA: diva2:142351
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
List of papers
1. A comparative analysis of 23 structures of the amyloidogenic protein transthyretin.
Open this publication in new window or tab >>A comparative analysis of 23 structures of the amyloidogenic protein transthyretin.
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2000 (English)In: J Mol Biol, ISSN 0022-2836, Vol. 302, no 3, 649-69 p.Article in journal (Refereed) Published
Abstract [en]

Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.

Keyword
Crystallography; X-Ray, Dimerization, Electrostatics, Humans, Hydrogen Bonding, Hydrogen-Ion Concentration, Models; Molecular, Molecular Sequence Data, Mutation, Prealbumin/*chemistry/genetics/metabolism, Protein Structure; Secondary, Protein Structure; Tertiary, Senile Plaques/*chemistry/genetics, Solubility, Solvents, Variation (Genetics), Water/metabolism
Identifiers
urn:nbn:se:umu:diva-13963 (URN)10986125 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2012-06-01Bibliographically approved
2. The β-strand D of transthyretin trapped in two discrete conformations
Open this publication in new window or tab >>The β-strand D of transthyretin trapped in two discrete conformations
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2004 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1700, no 1, 93-104 p.Article in journal (Refereed) Published
Abstract [en]

Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2004
Keyword
Crystallography, X-Ray, Dimerization, Humans, Hydrogen Bonding, Hydrogen-Ion Concentration, Models, Molecular, Mutation, Prealbumin, Protein Conformation, Protein Denaturation
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:umu:diva-13614 (URN)10.1016/j.bbapap.2004.04.004 (DOI)000222367400012 ()15210129 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2017-01-24Bibliographically 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: 2010-09-24Bibliographically approved

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