title: 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.
authors.affiliation: EMBL Hamburg Outstation, c/o DESY, Building 25a, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Cianci
authors.affiliation: Department of Food Science, University of Parma, Viale G. Usberti 23/a, 43124 Parma, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Folli
authors.affiliation: Shangai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China.
authors.forename: Francesco
authors.initials: F
authors.lastname: Zonta
authors.affiliation: Department of Life Sciences, University of Parma, Viale G. Usberti 23/a, 43124 Parma, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Florio
authors.affiliation: Department of Life Sciences, University of Parma, Viale G. Usberti 23/a, 43124 Parma, Italy.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Berni
authors.affiliation: Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35131 Padova , Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Zanotti
mesh_terms: D000686:Amyloidosis / Q000188:drug therapy / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D059808:Polyphenols / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D008024:Ligands; D059808:Polyphenols; D011228:Prealbumin
keywords: amyloidosis; fibrillogenesis inhibitors; negative cooperativity; protein misfolding; transthyretin; transthyretin stabilizers