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Structural evidence for asymmetric ligand binding to transthyretin
Michele Cianci1, Claudia Folli2, Francesco Zonta3
1EMBL Hamburg Outstation, c/o DESY, Building 25a, Notkestrasse 85, 22603 Hamburg, Germany.
Abstract:
Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.
Insights
Researchers discovered that transthyretin (TTR) has asymmetric binding sites for drugs, a finding that could lead to new combination therapies for TTR amyloidosis by targeting distinct sites.
Area of Science:
- Biochemistry
- Structural Biology
- Pharmacology
Background:
- Transthyretin (TTR) is an amyloidogenic protein implicated in TTR amyloidosis.
- Stabilizing TTR's native state is a therapeutic strategy for TTR amyloidosis.
- TTR possesses two thyroxine (T4) binding sites with negative binding cooperativity.
Purpose of the Study:
- To investigate the binding characteristics of natural polyphenols to TTR.
- To understand the structural basis of asymmetric ligand binding to TTR.
- To explore the therapeutic potential of TTR binding asymmetry.
Main Methods:
- X-ray crystallography was used to determine the structures of TTR complexed with pterostilbene, quercetin, and apigenin.
- Structural analysis was performed to identify differences between the two T4 binding sites.
- Molecular-dynamics simulations and competition binding assays were conducted to study binding in solution.
Main Results:
- Natural polyphenols (pterostilbene, quercetin) showed preferential binding to one TTR site, with lower occupancy in the second site.
- Structural analysis revealed subtle conformational differences between the two T4 binding sites.
- Molecular dynamics suggested more pronounced differences in solution, and competition assays confirmed distinct binding preferences for polyphenols versus T4.
Conclusions:
- TTR exhibits significant binding asymmetry for natural polyphenols and T4.
- This asymmetry is characterized by distinct binding affinities and site preferences.
- Exploiting this TTR binding asymmetry with combination drug therapy could enhance protein stabilization and treat TTR amyloidosis.
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