Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding of the ubiquitous enzyme Cu͞Zn superoxide dismutase (SOD). In contrast to other protein-misfolding disorders with similar neuropathogenesis, ALS is not always associated with the in vivo deposition of protein aggregates. Thus, under the assumption that all protein-misfolding disorders share at primary level a similar disease mechanism, ALS constitutes an interesting disease model for identifying the yet-mysterious precursor states from which the cytotoxic pathway emerges. In this study, we have mapped out the conformational repertoire of the apoSOD monomer through analysis of its folding behavior. The results allow us to target the regions of the SOD structure that are most susceptible to unfolding locally under physiological conditions, leading to the exposure of structurally promiscuous interfaces that are normally hidden in the protein's interior. The structure of this putative ALS precursor is strikingly similar to those implicated in amyloid disease.neurodegenerative disease ͉ protein folding ͉ transition state A n increasing number of severe neurodegenerative disorders have been linked to misfolding and subsequent aggregation of proteins (1). In some cases the protein aggregates deposit in the form of highly ordered amyloid fibrils (2), whereas in other cases they appear more granular or even amorphous (3). Even so, the main cause of neurotoxic function seems not to be the macroscopically deposited protein itself but rather a mysterious precursor species in the aggregation pathway (1, 4-6). In this perspective, it is notable that there are also disease cases where in vivo depositions of the disease-associated proteins are hard to find and sometimes are not even observable. Characteristic examples are found among the sporadic cases of CreuzfeldtJacob disease and amyotropic lateral sclerosis (ALS) (7). It is further apparent that the ALS-associated enzyme Cu͞Zn superoxide dismutase (SOD) has relatively low propensities to aggregate in vitro: SOD can be produced and analyzed by standard protocols despite being destabilized to the extent that it is half unfolded in physiological buffer at 25°C (8). For comparison there are few nondisease proteins that would resist aggregation under these conditions. The question then arises, do the disease cases with and without in vivo deposition of protein aggregates share at the primary level the same toxicity mechanism? We have previously observed that ALS-provoking SOD mutations produce a common shift of the folding equilibrium toward unfolded and partly folded monomers, implicating that the noxious side reaction emerges from the early folding events. The observation is in full agreement with a series of reports pointing at the immature apoSOD monomer as precursor for cytotoxicity (8)(9)(10)(11)(12)(13)(14)(15). Accompanying the shift of the folding equilibrium, the ALS-associated SOD mutations indicate a quantitative relation between decreased protein stability, net charge, and diseas...

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 M 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. functional evolution ͉ protein disease ͉ protein misfolding

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 motor-neuron disorder amyotrophic lateral sclerosis (ALS) 2 has been associated to more than 100 missense mutations in the radical scavenger copper/zinc superoxide dismutase (SOD1) (1). In analogy with several other neurodegenerative disorders (2), ALS seems associated with protein misfolding and aggregation (3-8). The principal support for such a disease mechanism is that the neural damage is accompanied by the accumulation of intracellular SOD1 deposits (9, 10). Even so, it is not the mature protein inclusions that seem to pose the main problem but rather some yet unidentified precursor accumulating in connection to the macroscopic aggregation process (11)(12)(13)(14)(15)(16). Consistent with such a scenario, transgenic mice suffer neuron stress long before any SOD1 inclusions can be detected (10,17,18). At the molecular level, studies of how the individual ALS-associated mutations affect the SOD1 molecule have further indicated protein stability as an important factor in the disease mechanism (19 -25); the more severe the destabilization caused by the mutation, the faster the disease progression (20). In this study, we shed further light on the disease-provoking properties of the SOD1 molecule by examining bioinformatically the chemical nature of 100 ALS-associated missense mutations. The results reveal an additional disease factor; the mutations have an overall tendency to decrease the net negative charge of the proteins. The negative charge repulsion that generally assures macromolecular solubility in vivo is diminished. This tendency is stronger for ALS mutations than in other hereditary protein disorders and cannot be discerned in the interspecies variability of the SOD1 sequence. In the few contrasting cases where the SOD1 mutations produce an increased repulsive charge, the side chain substitution tends also to be structurally obstructive and to occur in strictly conserved positions. That is, the opposed effect of increased repulsive charge is not sufficiently strong to compensate for the accompanying penalties in protein stability. The observation shows that the decreased net negative charge of SOD1 is a critical contributory factor to the ability of the mutation to cause neural damage. Taken together with the impact of decreased protein stability, the data also provide genetic s...

How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn(2+) first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn(2+) ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of ∼10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.