Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Abstract

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

Fig. 1.

Crystal structures and fibril morphologies of the apoSOD1 variants examined. The apoSOD1 monomer with intact C57–C146 crosslink (apoSOD1^C57–C146), the corresponding protein in TCEP with reduced C57 and C146 moieties (apoSOD1^SH SH), and the variant with loops IV (green) and VII (blue) removed by protein engineering (apoSOD1^ΔIV ΔVII). Structures constructed from PDB entry 2XJK. The EM micrographs of the respective protein fibrils, produced by agitation in pure pH 6.3 buffer at 37 °C. Scale bars are 200 nm.

Fig. 2.

Folding and fibrillation data. (A) Chevron plots of apoSOD1^C57–C146, apoSOD1^SH SH, and apoSOD1^ΔIV ΔVII showing fits of log k_f and log k_u [Eq. 8]. The curvatures in the unfolding limbs at high [urea] are due to transition-state shifts or native-state fraying and were excluded from the fits. The dotted line indicates the urea concentration where D reaches full occupancy. Units are in s^-1. (B) Concentration of D in the fibrillation assays as calculated from the chevron data of apoSOD1^ΔIV ΔVII. Total protein concentration is 25 μM, and units are in M. (C) Fibrillation lag times (τ_lag) of apoSOD1^ΔIV ΔVII vs. [urea] (open circles), derived from fibrillation time courses as described in SI Text, Analysis of Fibrillation Time Courses. The intrinsic [urea] dependence of log τ_lag was measured at full occupancy of the globally unfolded state (D) above 4 M urea. Subtraction of this [urea] dependence yields the direct relation between log τ_lag and log [D] (closed circles). Under these conditions, ∂ log τ_lag/∂[urea] = m_τlag = 0.13 ± 0.06. Units are in s. (D) Corresponding data and normalisation procedure for the elongation rate constants (ν_max) of apoSOD1^ΔIV ΔVII. ∂ log ν_max/∂[urea] = m_ν max = -0.17 ± 0.05. Units are in s^-1.

Fig. 3.

The fibrillation lag time (log τ_lag) and elongation rate constant (log ν_max) show linear dependencies on the logarithmized concentration of globally unfolded apoSOD1^ΔIV ΔVII (log[D]). The matching slopes of ∂ log τ_lag/∂ log[D] = -0.47 ± 0.03 and ∂ log ν_max/∂ log[D] = 0.56 ± 0.03 suggest a fragmentation-assisted mechanism according to Eqs. 3 and 5. Plots of log τ_lag vs. log ν_max, and τ_lag vs. ν_max (Inset). Data from urea titration (closed circles) and mutagenesis (open circles).

Fig. 4.

Schematic illustration of the bifurcated folding and fibrillation free-energy profiles of apoSOD1^ΔIV ΔVII. The reaction coordinate is solvent-accessible surface area, measured by the m-values in Table 1 and SI Text, Fibrillation m-values. The profiles indicate the case where the folding and fibrillation transition states (‡^folding and ‡^elongation) are placed symmetrically. For simplicity, the stabilities of the fibrillar state (A), the unfolded monomer (D) and the folded protein (N) are normalized, and barrier heights are not to scale. I* is the high-energy intermediate observed by NMR at 0 M urea (16, 23).

Fig. 5.

Examination of the apoSOD1^ΔIV ΔVII fibrillation kinetics by E-scan under fully denaturing conditions at 5 M urea. (A) Histograms of mutant lag times (τ_lag) fitted by non-normalized gamma distributions (Table 2). Pseudo-wild type (black) and mutants (red and green). (B) Sequence positions of E mutations. (C) Structural positions of E mutations (orange). Positively and negatively charged side chains are shown in blue and red, respectively.

Fig. 6.

Correlations between fibrillation lag times (τ_lag) and the parameters net charge and hydrophobicity change. (A) τ_lag shows an overall linear dependence on net charge, consistent with the general behaviour of protein-protein interactions, according to Scheme 2 (34). ApoSOD1^ΔIV ΔVII (-3), E-scan mutants (-4 to -6), and apoSOD1^C57–C146/apoSOD1^SH SH (-8). (B) Plot of τ_lag for the apoSOD1^ΔIV ΔVII mutants with normalized net charge of -5 against change in local hydrophobicity (Table 2). The outlier is the β1 mutation V5E/V7E, which is unique by having no effect on τ_lag. The R-value of 0.85 is without the β1 measurement.