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We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in copper/zinc superoxide dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins' net repulsive charge. For each SOD1 monomer this charge is normally -6. Because biomolecules as a rule maintain net negative charge to assure solubility in the cellular interior, the result lends support to the hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of the preferential reduction of repulsive charge is higher in SOD1-associated ALS than in other inherited protein disorders.

The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn 2+ loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a nonnative Zn 2+ ion with μM affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn 2+ ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features.

Amyotropic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding and aggregation of the homodimeric enzyme superoxide dismutase (SOD1). In contrast to the precursors of other neurodegenerative diseases, SOD1 is a soluble and simple-to-study protein with immunoglobulin-like structure. Also, there are more than 120 ALS-provoking SOD1 mutations at the disposal for detailed elucidation of the disease-triggering factors at molecular level. In this article, we review recent progress in the characterization of the folding and assembly pathway of the SOD1 dimer and how this is affected by ALS-provoking mutations. Despite the diverse nature of these mutations, the results offer so far a surprising simplicity. The ALS-provoking mutations decrease either protein stability or net repulsive charge: the classical hallmarks for a disease mechanism triggered by association of non-native protein. In addition, the mutant data identifies immature SOD1 monomers as the species from which the cytotoxic pathway emerges, and point at compromised folding cooperativity as a key disease determinant. The relative ease by which these data can be obtained makes SOD1 a promising model for elucidating also the origin of other neurodegenerative diseases where the precursor proteins are structurally more elusive.