Abstract
The structures and stabilities of transthyretin tetramers and their complexes with tolcapone or tafamidis bound at the T4-site, in aqueous solutions without and with 8 M urea, are revealed using SEC-SWAXS and NMR. Human transthyretin (TTR) is a homotetrameric protein involved in transporting thyroxine (T4) and retinol-binding protein within serum and cerebrospinal fluid. The disassociation of TTR’s tetrameric structure can lead to the formation of biologically toxic TTR amyloid fibrils. Tolcapone, a small molecule currently under clinical trial, has shown potential as a TTR stabilizer and may act as an alternative to tafamidis, the conventional therapeutic agent used to prevent TTR dissociation. Using size-exclusion-chromatography-based small- and wide-angle X-ray scattering (SEC-SWAXS) complemented by nuclear magnetic resonance (NMR) spectroscopy, this study reveals the solution conformations of Apo-TTR and TTR complexed with tolcapone and tafamidis. Our results indicate that both compounds can bind similarly to the two T4 sites of TTR, leading to a small increase in the radius of gyration from 24.3 ± 0.1 Å (Apo-TTR) to 25.8 ± 0.1 Å. Consequently, both compounds largely stabilize the TTR against dissociation, denaturation and oligomerization up to 8 M urea, whereas Apo-TTR starts to denature at this concentration and forms larger oligomers at 8 M urea. Additionally, under a reduced TTR–drug mixing ratio of 1:1, which targets only one T4 site, tafamidis more effectively stabilizes the TTR tetrameric conformation at 8 M urea, a difference attributed to its higher affinity for the first T4 site. These results illustrate an effective strategy for investigating protein–drug interactions by examining the solution conformations of protein–drug complexes under physiological conditions, providing structural hints to the design of therapeutic agents targeting TTR.