Two distinct aggregation pathways in transthyretin misfolding and amyloid formation

Abstract

Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2-5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.

Keywords: Amyloid formation; Misfolding; Solid-state NMR; Transthyretin.

Figure 1.

A schematic diagram for the two distinct misfolding and amyloid formation pathways of TTR at the two different acidic conditions (pH 2.4 and 4.4) along with 2D ¹H/¹⁵N HSQC NMR spectra and TEM images. A very low protein concentration of 0.15 mg/ml was used for the 2D ¹H/¹⁵N HSQC NMR experiments to ensure TTR exist as monomeric forms. The TEM images were obtained from the TTR samples (0.2 mg/ml and 10 mg/ml) incubated for 1 day and one month at pH 4.4 and 2.4, respectively. The different protein concentrations and incubation times were used because of the different aggregation kinetics. At pH 2.4, a much higher protein concentration was required to obtain amyloid precipitates due to the much slower aggregation kinetics than those at pH 4.4.

Figure 2.

Selective labeling schemes for the solid-state NMR experiments. The ¹³CO (green) and ¹³Cα (red) carbons of the amino acids are labeled if the internuclear distance in the native TTR tetramer is between 4 and 6 Å.

Figure 3.

(a) Overlaid 2D PDSD spectra of the amyloid-4 with ¹³CO-Leu/¹³Cα-Met (red; labeled on DA), and ¹³CO-Leu/¹³Cα-Tyr (black; labeled on AGH) TTR. (b) Overlaid 2D PDSD spectra of the amyloid-2 with the same labeling schemes in a. (c) 2D PDSD spectrum of the amyloid-2 with ¹³CO-Val/¹³Cα-Tyr labeled on GH strands. The 1D slice in Figure 3a and 3b was drawn for the L/M spin pairs. The NMR spectra were obtained with a mixing time of 500 ms at a ¹H frequency of 830 MHz. The L/Y and L/M spin pairs in the amyloid-4 state were unambiguously assigned in our previous solid-state NMR studies. [17] In order to confirm that the cross-peaks originate from the spin pairs within the TTR monomer, the 2D PDSD experiments were conducted on the mixture of singly ¹³CO- and ¹³Cα- labeled TTRs (1:1 ratio, for example a mixture of ¹³CO-Leu-TTR and ¹³Cα-Tyr-TTR) and no cross-peak was observed from the mixture. * denotes spinning sidebands.

Figure 4.

2D PDSD spectra of the two amyloid states for the labeling schemes shown in Figure 2. (a) ¹³CO-Ile/¹³Cα-Ala for the EF strands. (b) ¹³CO-Ile/¹³Cα-Val for the BE strands. (c) ¹³CO-Gly/¹³Cα-Val for the CB strands. The spin pairs in the amyloid-4 state were unambiguously assigned in our previous solid-state NMR studies. [17]

Figure 5.

Aggregation kinetics of the WT and mutant forms of TTR measured at pH 4.4 (a) and 2.4 (b). Optical density at 400 nm was monitored at pH 4.4, and dynamic light scattering was used to probe aggregation at pH 2.4. The scattering intensity at 100 μs in the scattering profile shown in Figure S8 was plotted and compared for the WT and mutant forms of TTR. Errors in the experimental measurements were mostly smaller than the size of the markers.