A relationship between the transient structure in the monomeric state and the aggregation propensities of α-synuclein and β-synuclein

Abstract

α-Synuclein is an intrinsically disordered protein whose aggregation is implicated in Parkinson's disease. A second member of the synuclein family, β-synuclein, shares significant sequence similarity with α-synuclein but is much more resistant to aggregation. β-Synuclein is missing an 11-residue stretch in the central non-β-amyloid component region that forms the core of α-synuclein amyloid fibrils, yet insertion of these residues into β-synuclein to produce the βSHC construct does not markedly increase the aggregation propensity. To investigate the structural basis of these different behaviors, quantitative nuclear magnetic resonance data, in the form of paramagnetic relaxation enhancement-derived interatomic distances, are combined with molecular dynamics simulations to generate ensembles of structures representative of the solution states of α-synuclein, β-synuclein, and βSHC. Comparison of these ensembles reveals that the differing aggregation propensities of α-synuclein and β-synuclein are associated with differences in the degree of residual structure in the C-terminus coupled to the shorter separation between the N- and C-termini in β-synuclein and βSHC, making protective intramolecular contacts more likely.

Figure 1

Alignment of the amino acid sequences of αS, βS, and βS_HC. Amino acids are colored according to the chemical nature of their side chains. The region shaded in Cambridge blue indicates the 11 residues from αS that were inserted into βS to form βS_HC.

Figure 2

Intensity ratios I_ox/I_red for each spin-label position for (A) αS,^, (B) βS, and (C) βS_HC. The experimental data are shown as black bars, and the I_ox/I_red values calculated from the random coil ensemble are plotted as thick red lines. PRE–NMR experiments were conducted on 100 μM uniformly ¹⁵N-labeled protein with MTSL attached in 10 mM sodium phosphate (pH 7.4), 100 mM NaCl, and 10% D₂O at 10 °C. The experimental I_ox/I_red values are those processed for use in the simulations (see Methods); thus, any I_ox/I_red of <0.15 or >0.85 has been set to 0.15 or 0.85, respectively. If no bar is present, then either I_ox/I_red was not measured for this residue or it was discarded because of an uncertainty of >10%.

Figure 3

R_g probability distributions for (A) αS, (B) βS, and (C) βS_HC. The random coil ensembles (see the text for a definition) are colored black, and the ensembles calculated using PRE-RMD are colored red. Representative structures are shown for various values of R_g. The R_g distributions are shown rather than the R_h distributions because the former are faster to calculate, but the R_h distributions are similar. (D) Distributions of the difference between the random coil and PRE-RMD ensemble R_g probabilities [Δp(R_g) = p(R_g^random coil) – p(R_g^PRE-RMD)].

Figure 4

Comparison of experimentally measured^, and calculated NMR observables for (A and D) αS, (B and E) βS, and (C and F) βS_HC. (A–C) ³J_HNHa couplings (black) measured experimentally, (red) calculated from the PRE-RMD ensembles, and (green) calculated from random coil ensembles. (D–F) Amide N–H RDCs measured experimentally in (black) C8E5/octanol or (blue) Pf1 bacteriophage, (red) calculated from the PRE-RMD ensembles, and (green) calculated from random coil ensembles.

Figure 5

Distance comparison (DC) maps for the (A) αS, (B) βS, and (C) βS_HC ensembles determined by PRE-RMD. The top half shows the full DC map, whereas the bottom half shows only the scaled distances that are less than 75% of that expected for a random coil polymer and occur between pairs of oppositely charged residues. The same color scale is used for all the DC maps to aid comparisons.

Figure 6

Free energy landscapes of structural ensembles determined for (A) αS, (B) βS, and (C) βS_HC ensembles. The free energy is defined as F(R_g,SASA) = −ln p(R_g,SASA). Examples of structures found at various points on each landscape are given, and the position of the experimental micelle-bound structure of αS and a homology model of βS based on the αS structure are indicated by filled cyan circles.

Figure 7

Aggregation propensity, Z_agg^prof, predicted using the Zyggregator algorithm for (black, solid) αS, (red, solid) βS, and (green, dashed) βS_HC. The residue numbers and gaps correspond to the sequence alignment shown in Figure 1. The gray line at Z_agg^prof = 1 indicates the threshold for classifying a sequence as being aggregation prone; regions exhibiting Z_agg^prof values greater than this are considered to be aggregation prone.