Structural evidence for asymmetric ligand binding to transthyretin

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.

Evaluating the effect of mutations and ligand binding on transthyretin homotetramer dynamics

“…Besides, fluorescence experiments shows that resveratrol metabolites, like resveratrol-3-O-sulfate, have lower affinities for TTR than that of resveratrol[51]. In good agreement with that, we obtain (T4)< (resveratrol)< (resveratrol-3-O-sulfate).…”

“…Besides, experiments reveal that, at equal concentrations, quercetin is able to displace pterostilbene[51], in agreement with the lower value of for quercetin with respect to pterostilbene.…”

“…Performing competition binding assays, it has been shown that the polyphenols pterostilbene and quercetin have different preferential binding sites in TTR than the one of T4[51]. Besides, experiments reveal that, at equal concentrations, quercetin is able to displace pterostilbene[51], in agreement with the lower value of for quercetin with respect to pterostilbene.…”

“…Both modes imply relative motions mainly localized on EF-loop, FG-loop and BC-loop affecting only one of both dimer-dimer interfaces. Previous studies[51] show that TTR ligands present different abilities to saturate the two T4 binding sites, leading to the presence of a main binding site with clear ligand affinity and a second minor site with a much lower ligand affinity. Our results complement these previous structural evidence for asymmetric TTR-ligand binding related to its negative binding cooperativity[61], [62].…”

“…While neither pterostilbene nor quercetin is able to displace TTR-bond T4, the polyphenol apigenin displaces it but only at very high concentrations[51]. These results indicate that apigenin presents lower affinity than T4.…”