A molecular mechanism for transthyretin amyloidogenesis

Abstract

Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein.

Fig. 1

Structure of human TTR and experimental studies of TTR stability. a–c Crystallographic structure of WT-TTR (PDB-ID 4PVM). Tafamidis is shown in green (by superimposition of PDB-ID 6FFT), the S52 amino acid in red, and T119 in blue. d Scheme of the native MS experiment: 4H (hydrogenated) and 4D (deuterated) tetramers are mixed in equal parts; following dissociation, four hybrid tetrameric species may form by the assembly of monomeric and dimeric species. e–h MS result for the subunit exchange between deuterated S52P and hydrogenated WT, S52P, T119M, and S52P bound to tafamidis, respectively. Changes in relative abundance of homo- and hetero-tetrameric species over the course of 11 days are shown, along with an estimate of the of their association/dissociation rates (uncertainties are one standard deviation of the posterior densities)

Fig. 2

Relative stability of mutant tetramers, dimers, and monomers, with respect to WT-TTR. ΔΔG_(tetr) is the stability of the mutant tetramers with respect to WT-TTR for the process of dissociation to dimers; ΔΔG_(dime) represents the relative stability of the mutant dimers when they dissociate to monomers; ΔΔG_(mono) indicates the relative stability of the mutant monomers for unfolding; ΔΔG_(unf) is the relative stability of the mutant tetramers for the overall processes of tetramer dissociation and unfolding. The uncertainties shown are one standard error of the mean, obtained from ten repeated calculations

Fig. 3

Neutron structures and molecular dynamics results. a In the T119M mutant (as well as in the WT; not shown), the Ser52 residue forms two hydrogen bonds (yellow dash lines) with the Ser50 residue. The distance between Ser50-C_α and the amide-N of Ser52 is 4.2 Å (grey), and it is the same in the WT (not shown). b In the S52P mutant, due to the absence of the  hydrogen bonds between Pro52 and Ser50, the distances between Ser50-C_α and the amide-N of Pro52 is longer (4.6 Å), creating a looser CD loop. c Binding of tafamidis (3MI) results in a change of orientation for residue Thr119 (cyan, before binding; green, after binding). In the S52P/tafamidis complex, the hydroxyl side chain of Thr119 forms a hydrogen bond to a water molecule. d The water molecule in the S52P/tafamidis complex occupies the same position as the side chain of Met119 residue (light orange) in the non-pathogenic T119M mutant. The binding site between chain B and B′ is shown. The blue and magenta mesh show the 2F_o-F_c neutron scattering length density map and the 2F_o-F_c X-ray electron density map, respectively. Maps were contoured at 1.5 σ. e–g Loss of native fold and contacts in WT-TTR during high-temperature MD simulations with the Amber99sb*-ILDNP force field; shown are the mean and its standard error from 10 simulation repeats (results for Charmm36 are in Supplementary Figure 6). e Degree of disruption of the monomer fold, the monomer–monomer interface, and the dimer–dimer interface, when simulating the WT tetramer over the course of 100 ns. f Degree of unfolding of TTR monomers as part of a tetramer, dimer, or monomer in solution. g Degree of disruption of the monomer–monomer interface when simulating the dimer and the tetramer. h Protein regions in the PLS-FMA mode contributing most to the change in the fraction of native contacts for the high-temperature simulations

Fig. 4

Proposed model of mutational effect on TTR stability. Shorter arrows indicate a lower propensity to association/folding. Red arrows reflect the qualitative change in reaction rates for the mutants with respect to WT-TTR. a The S52P mutation increases the likelihood of partial and full unfolding of TTR monomeric units. This, in turn, also leads to a lower stability of TTR multimeric assemblies; hence partially unfolded dimers and monomers, and fully unfolded monomers, are generated at a higher rate. Some or all of these species are then removed from solution via the formation of amyloid fibrils. b In contrast to S52P, the T119M mutation stabilises the folded monomeric forms of TTR. Most importantly, this mutation has a large stabilising effect on TTR tetramers, which are believed not to form fibrils. Furthermore, it is proposed that the higher stability of the tetramer also has the effect of further stabilising the monomer fold