The Fluorescent Amyloid Ligand X34 Binding to Transthyretin (TTR) Tetramer and Fibrils: FRET and Binding Constants of a Sequential Two-step Process

The amyloidogenic homotetrameric plasma protein transthyretin (TTR) has an affinity for bicyclic small molecule ligands in its two thyroxine (T4) binding sites. We have shown that native tetrameric TTR binds to amyloid ligands based on the trans-stilbene scaffold. The fluorescent Congo-red analogue, X34, is a symmetric bi-trans-stilbene that contains two salicylic acid motifs. We used fluorescence spectroscopy methods to interrogate X34 binding to the TTR tetramer and fibril. We discovered two binding sites in both TTR forms by tryptophan FRET, ligand self-quenching, Stern-Volmer plots and binding curves, for the latter including the competitive ligand diflunisal. X34 binds with the similar affinity as diflunisal in the first binding site (K_d1=150 nM), and negative cooperativity renders the binding to the second site with lower affinity very similar compared to diflunisal (K_d2= 1.1 μM). This behavior is coherent with the salicylic acid moiety of diflunisal binding into the binding pocket of TTR (reverse mode). Interestingly X34 binding to TTR fibrils was also well fitted to two binding sites, however with overall lower affinity (K_d1=1.2 μM; K_d2=2.1 μM) compared to binding to the native tetramer. X34 fluorescence when bound to TTR-fibrils was significantly blue shifted compared to binding to the TTR-tetramer.