Zn2+-Induced Dissociation of Transthyretin Tetramers Generates Toxic, Seeding-Active Misfolded Oligomers.

The aggregation of transthyretin (TTR) is a hallmark of ATTR amyloidosis. Accumulating evidence suggests that metal ions, particularly Zn2+, promote TTR aggregation; however, the molecular mechanism underlying Zn2+-induced misfolding remains poorly understood. Here, we present a comprehensive biophysical characterization of Zn2+-induced misfolding of wild-type transthyretin (TTRwt) under physiologically relevant conditions. Two-dimensional 1H/15N HSQC NMR analyses reveal that Zn2+ binding induces millisecond-time scale conformational exchange that destabilizes the native tetramer. These structural and dynamic perturbations promote the formation of small, toxic oligomeric species capable of self-propagation through the seeding of native TTR. Comparative structural analyses using solid-state NMR also show that Zn2+-induced oligomers adopt broadly native-like secondary structures, but exhibit distinct non-native molecular conformations. The early stages of misfolding were further investigated using 19F NMR of TTRwt containing fluorinated tryptophan residues. Notably, 19F NMR not only reports on Zn2+ binding but also enables direct detection of a transient monomeric intermediate generated upon tetramer dissociation, a key species in the amyloidogenic cascade. Together, these findings define the structural and dynamic basis of Zn2+-mediated TTR misfolding and establish combined solution and solid-state NMR approaches as powerful tools for probing metal-induced misfolding landscapes in TTR and related amyloidogenic proteins.