Kinetic analysis of the multistep aggregation pathway of human transthyretin

Abstract

Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a ¹⁹F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct ¹⁹F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time ¹⁹F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119M) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119M remains resistant to tetramer dissociation and aggregation.

Keywords: aggregation kinetics; amyloidogenic protein; hydrophobic interaction; low-population intermediate; real-time NMR.

Fig. 1.

The TTR^F tetramer showing the BTFA labels at S85C and the mutation sites. (A) Modeled TTR^F tetramer is based on PDB 1BMZ (51), where BTFA is conjugated at S85C with Cys side chain atoms shown in gray spheres and the three fluorine atoms highlighted in magenta. The four protomers are colored individually. Cα locations of pathogenic (V30M, L55P, and V122I) and protective (T119M) mutations are shown in cyan spheres for the yellow protomer. The Cα of F87 from the pink protomer is drawn as a red sphere. (B) Expanded view of the dashed region in A. The side chain heavy atoms of V30, L55, T119, and V122 from the yellow protomer are shown as cyan spheres, and the side chain heavy atoms of F87 from the pink protomer are shown as red spheres. The chemical structure of BTFA conjugated to the thiol in the Cys side chain is shown at the bottom right.

Fig. 2.

Monitoring aggregation kinetics of TTR^F at pH 4.4 by integrating ¹⁹F-NMR and turbidity measurements. (A) Contour plot of time-dependent ¹⁹F-NMR spectra of TTR^F at 298 K with a time resolution of 1 h. Relative intensity from high to low is colored from red to blue. A representative one-dimensional ¹⁹F-NMR spectrum recorded from the 9th to 10th hour is shown below with tetramer (T) and intermediate (I) peaks labeled. The ¹⁹F-NMR peak areas (left black axis) and optical density at 330 nm (OD₃₃₀, right orange axis) plotted as a function of aggregation time at (B) 298 K, (C) 310 K, and (D) 277 K. The peak area data points are colored blue for tetramer (T) and red for intermediate (I). Green points represent the missing signal amplitude for the ensemble of aggregates (A), derived by subtracting the sum of (T + I) from 1. The maximal OD₃₃₀ (see SI Appendix, Fig. S8B, for a full range of time points) is normalized to the maximum of the A signal for convenience of comparison. The black lines show the fits of the NMR kinetic data based on Scheme 1. The rate of formation of higher order aggregates at 310 K was approximated by fitting the normalized OD₃₃₀ data to a single exponential function (orange line). The orange connecting lines in OD₃₃₀ at 298 and 277 K are for eye guidance. Error bars in OD data represent 1 SD from three independent measurements in C but are not shown in B and D because they are smaller than the size of data markers.

Scheme 1.

Fig. 3.

Temperature dependence of aggregation kinetics of pathogenic and protective mutants at pH 4.4. The ¹⁹F-NMR contour plots of (A) V30M^F, (B) L55P^F, (C) V122I^F, and (D) T119M^F are shown at 298 K. A representative ¹⁹F NMR spectrum recorded from the 9th to 10th hour is shown below each contour plot (see SI Appendix, Figs. S3 and S4, for contour plots at 310 and 277 K, respectively). The time-dependent changes in the tetramer (T) resonance areas of (E) V30M^F, (F) L55P^F, (G) V122I^F, and (H) T119M^F are plotted at three temperatures. For each plot, the first data point is normalized to 1. Exponential fits are in black solid lines (see SI Appendix, Fig. S9, for fitting details).

Fig. 4.

Comparison of kinetic parameters for TTR^F and F87A^F. (A) Tetramer dissociation rate k₁. (B) Monomer tetramerization rate k₋₁. (C) Forward oligomerization rate k₂. (D) Reverse oligomerization rate k₋₂. Uncertainty was estimated as 1 SD from 50 bootstrap datasets.

Fig. 5.

Comparison of the populations of the intermediates formed by TTR^F, V122I^F, and F87A^F at pH 4.4. (A) Time-dependent ¹⁹F-NMR contour plot of F87A^F at 298 K. A representative spectrum recorded from the 9th to 10th hour is shown below. Compared with TTR^F (Fig. 2A) and V122I^F (Fig. 3C), the intermediate population predominates over tetramer during this time period. (B) The relative population of the intermediate at steady state (I_steady) for TTR^F (black), V122I^F (cyan), and F87A^F (red). van’t Hoff plot for (C) the apparent tetramer–intermediate equilibrium constant (K_app,1 = [I]/[T]) and (D) the apparent intermediate–aggregate equilibrium constant (K_app,2 = [A]/[I]) for TTR^F, V122I^F, and F87A^F. In C and D, black lines are linear fits to the van’t Hoff equation, and dashed lines denote zero. Uncertainties in B–D were estimated as 1 SD from 50 bootstrap datasets. Some uncertainties in B and D are comparable to or smaller than the size of data marker.

Fig. 6.

Schematic free energy level diagram at pH 4.4 of TTR^F (black), V122I^F (cyan), and F87A^F (red) at (A) 310 K, (B) 298 K, and (C) 277 K based on Scheme 1. The apparent free energy of the I state is set to 0 as reference for all three temperatures (dashed line). The transition states between T and I and between I and A are designated 1* and 2*, respectively.