Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state

Abstract

More than 100 point mutations of the superoxide scavenger Cu/Zn superoxide dismutase (SOD; EC ) have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, these mutations are scattered throughout the protein and provide no clear functional or structural clues to the underlying disease mechanism. Therefore, we undertook to look for folding-related defects by comparing the unfolding behavior of five ALS-associated mutants with distinct structural characteristics: A4V at the interface between the N and C termini, C6F in the hydrophobic core, D90A at the protein surface, and G93A and G93C, which decrease backbone flexibility. With the exception of the disruptive replacements A4V and C6F, the mutations only marginally affect the stability of the native protein, yet all mutants share a pronounced destabilization of the metal-free apo state: the higher the stability loss, the lower the mean survival time for ALS patients carrying the mutation. Thus organism-level pathology may be directly related to the properties of the immature state of a protein rather than to those of the native species.

Fig 1.

Crystal structure of homodimeric Cu/Zn SOD from human (PDB code ), showing the positions of the Cu (green) and the Zn (gray) ions and the side-chain positions mutated in this study (red).

Fig 2.

CD-monitored GdmCl titrations of SOD^WT. θ is in units of 10³ deg⋅cm²⋅dmol⁻¹. (Upper) Unfolding transitions of apo-SOD^WT (▪) and holo-SOD^WT (•) monitored by CD at 216 nm, under conditions of reduced cysteines. The curves are fits of Eq. 1. The apoprotein shows a significantly lower midpoint and higher m value than the holoprotein (Table 1). (Lower) Unfolding transition of holo-SOD^WT (•) at 230 nm, and analogous data from a standard protein preparation with heterogeneous metal content (○) under conditions that promote formation of disulfide crosslinks. The latter transition reveals clearly the presence of apo-SOD and other nonnative species that are not discerned in fresh homogenous preparations.

Fig 3.

Unfolding transitions for the ALS-associated mutants (open symbols) and SOD^WT (filled symbols) measured by CD. The GdmCl data were monitored at 216 nm and the urea data were monitored at 230 nm. The mutants SOD^D90A, SOD^G93A, and SOD^G93C show more pronounced stability effects on the apoprotein (□ and ▪) than on the holoprotein (○ and •), whereas the disruptive mutations SOD^A4V and SOD^C6F show radical perturbations of both the apo- and holoprotein. The control mutation SOD^C6A shows no marked effect on either species.

Fig 4.

Histogram of the stability loss (ΔMP) upon mutation of SOD. The ALS-associated mutations show a common destabilization of the apoprotein (red) even as the effect on holo-SOD varies (blue). The experiments were done at different redox potentials to distinguish possible artifacts from erroneous disulfide bridges. □, 0 M TCEP; , 50 μM TCEP, , 100 μM TCEP; and , 500 μM TCEP.

Fig 5.

Far-UV CD and absorbance spectra of the various SOD species. θ is in units of 10³ deg⋅cm²⋅dmol⁻¹. The only mutations indicative of structural perturbations are SOD^A4V and SOD^C6F. (a) CD spectra of holo-SOD^WT (•), apo-SOD^WT (○), and holo-SOD^WT denatured in 6 M GdmCl (▪). (b) Absorbance spectra of holo-SOD^WT (•) and apo-SOD^WT (○). (c) CD spectra of the holo-SOD mutants: •, wild type; ♦, A4V; ▪, C6A; ⋄, C6F; ×, D90A; □, G93A; and ▵, G93C. (d) CD spectra of the apo-SOD mutants: ○, wild type; ♦, A4V; ▪, C6A; ⋄, C6F; ×, D90A; □, G93A; and ▵, G93C.

Fig 6.

Plot of mean survival time after ALS diagnosis versus stability loss of apo-SOD (ΔMP) upon mutation. ΔMP is the average value from the different reducing conditions in Table 1. The fitted line is y = 25.9 years − 28x; R = 0.91. A tentative interpretation would be that the intersect of 25.9 years corresponds to the survival time of (sporadic) ALS cases with no SOD mutation, but with genetic factors that are otherwise identical to those of the patient group in Table 1.