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To this end, we identified two novel mutants, A89R and K128N, which were predicted to be highly destabilizing or stabilizing, respectively.

Conversely, we asked whether a mutant could be designed de novo that would further stabilize SOD1 and render it more resistant to misfolding pathology. In light of this evidence, we were inspired to consider if a de novo non-fALS SOD1 mutation could be rationally designed to present phenotypes similar to SOD1-fALS mutants in commonly used experimental SOD1-related fALS models. The variable effects of different mutations on these biophysical components of SOD1 stability and aggregation have been suggested to be responsible for the variable patient survival times in SOD1-fALS( 10, 12- 14). Indeed, SOD1-related fALS mutations are reported to decrease the stability of dimer formation( 7, 8), monomer folding( 9, 10), and metal binding( 11). Loss of any of these factors increases the likelihood of SOD1 misfolding into a toxic disease state due to a decrease in stability and invariable formation of toxic aggregates( 6). A properly folded monomer binds one zinc and one copper atom, and contains an intramolecular disulfide bond, all of which are important for the stability of the protein( 4). When natively folded, SOD1 is a 32 kDa homodimer that catalyzes the dismutation of oxygen radicals to less harmful molecular oxygen and hydrogen peroxide( 4). The ubiquity of disease-associated mutations (currently over 160( 5)) throughout the primary sequence of SOD1 suggests a physico-chemical origin for SOD1-related fALS. Most cases of ALS cases are sporadic with no consistently identified genetic mutation, however, a significant fraction of familial ALS (fALS) cases are due mutations in Cu,Zn superoxide dismutase (SOD1)( 2- 4). We propose a mechanism for this rescue, which may provide an avenue for therapeutic intervention.Īmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons in the motor cortex, brainstem and spinal cord, with a lifetime risk of about 1:400( 1). In fact, it was able to rescue the ALS phenotype caused by mutant SOD1. We found that the unstable mutant was toxic, and induced a severe ALS phenotype in zebrafish the predicted stable mutant, on the other hand, behaved even better than WT. We then tested these mutants using in vitro, cell-based, and in vivo zebrafish models. We sought to pursue this thread using a synthetic biology approach, where we designed two de novo mutations, one stabilizing and one destabilizing, as predicted using computational molecular dynamics simulations. Significance Mutations in the protein superoxide dismutase cause ALS, and many of these mutants have decreased folding stability. The demonstrated success of these first principles calculations to predict the physical properties of SOD1 mutants holds promise for rationally designed therapies to counter the progression of ALS. Consistent with this prediction, co-injection of K128N and A4V into zebrafish shows profound rescue of motor neuron pathology. Moreover, K128N is predicted computationally to form an unusually stable heterodimer with the familial ALS mutant A4V. The experimental tests validated the theoretical predictions: A89R is an unstable, highly-deleterious mutant, and K128N is a stable, non-toxic mutant.

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We subjected these in silico designed mutants to a series of experimental tests, including in vitro measures of thermodynamic stability, cell-based aggregation and toxicity assays, and an in vivo developmental model of zebrafish motor neuron axonopathy. We address this problem here by computationally designing two de novo mutants, A89R and K128N, which were predicted theoretically to be either significantly destabilizing or stabilizing respectively. The underlying physical causes of SOD1-related ALS are still not well-understood.

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