Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity

Abstract

Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance-presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30MT119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.

Figure 1

The structure of tetrameric TTR, showing the location of the point mutations that change the kinetics and/or thermodynamics of partial denaturation and influence amyloidogenicity and disease phenotype (mutations identified in only one subunit). The V30M TTR mutation is located in the hydrophobic core, whereas the L55P substitution is located in β-strand D. The V122I TTR mutation is close to the C terminus and the dimer/dimer interface region of TTR. T119M contributes to the dimer/dimer interface and comprises the thyroxine-binding sites. The T119M TTR mutation appears to stabilize the quarternary structure and dramatically slows the rate of tetramer dissociation, enabling it to function as a transsuppressor of misfolding (20), apparently by increasing the hydrophobic surface area buried at the dimer/dimer interface (34).

Figure 2

Evaluation of the stability of TTR sequences as a function of urea concentration. WT, filled triangles; V30M, open squares; L55P, filled circles; V122I, open circles; T119M, open triangles. (a) Tetramer dissociation curve measured by resveratrol binding (96-h incubation). (b) Tertiary structure unfolding curve measured by intrinsic tryptophan fluorescence changes.

Figure 3

TTR tetramer dissociation rates as a function of urea concentration (Fig. 2 symbols apply). (a) Unfolding time course measured by tryptophan fluorescence provides the rate of TTR tetramer dissociation in 6.0 M urea. Inset shows the rapid unfolding of monomeric TTR in 5.0 M urea measured by stopped-flow fluorescence. (b) The logarithm of the rate of tetramer dissociation, lnk_diss (k_diss in h⁻¹) plotted as a function of urea concentration. The lnk_diss vs. urea concentration plot is linear, allowing extrapolation to 0 M urea.

Figure 4

TTR fibril formation time courses detected by turbidity at 400 nm (OD⁴⁰⁰). (a) Amyloid fibril formation mediated by partial acid denaturation of TTR (0.20 mg/ml) in acetate buffer (pH 4.4, 37°C, Fig. 2 symbols apply). (b) TTR (0.10 mg/ml) amyloid fibril formation enabled by MeOH-induced denaturation [50% (vol/vol) in Tris buffer (pH 7.0, 25°C)].

Figure 5

The kinetics of WT (filled triangles) and T119M TTR (open triangles) reconstitution (folding and reassembly) monitored by resveratrol fluorescence (monitors tetramer formation). A 10-fold dilution of urea unfolded TTR (8 M) to a final TTR concentration of 1.8 μM_tetramer initiates reconstitution (final urea concentration = 1.0 M). Inset shows the complete trace for the very slow T119M reassembly time course. The rate constants (extrapolated to 0 M urea) for the fast phase are 0.294 ± 0.014 s⁻¹ (WT) and 3.16⋅10⁻³ ± 1.65⋅10⁻³⋅s⁻¹ (T119M). For the slow phase, the rate constants are 6.24⋅10⁻² ± 0.62⋅10⁻²⋅s⁻¹ (WT) and 2.76⋅10⁻⁴ ± 2.3⋅10⁻⁴⋅s⁻¹ (T119M). Therefore, the reassembly rate of T119M (extrapolated to 0 M urea) is 93-fold slower (fast phase) and 226-fold slower (slow phase) relative to WT.