Differentiating the solution structures and stability of transthyretin tetramer complexed with tolcapone and tafamidis 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.