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Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD12SH, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD12SH have shown that the ground state interconverts with a series of sparsely populated and transiently formed conformers, some of which have aberrant nonnative structures. Here, we study seven disease mutants of apoSOD12SH and characterize their free energy landscapes as a first step in understanding the initial stages of disease progression and, more generally, to evaluate the plasticity of low-lying protein conformational states. The mutations lead to little change in the structures and dynamics of the ground states of the mutant proteins. By contrast, the numbers of low-lying excited states that are accessible to each of the disease mutants can vary significantly, with additional conformers accessed in some cases. Our study suggests that the diversity of these structures can provide alternate interaction motifs for different mutants, establishing additional pathways for new and often aberrant intra- and intermolecular contacts. Further, it emphasizes the potential importance of conformationally excited states in directing both folding and misfolding processes.

SUBMITTER: Sekhar A

PROVIDER: S-EPMC5111666 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Sekhar Ashok A Rumfeldt Jessica A O JAO Broom Helen R HR Doyle Colleen M CM Sobering Ryan E RE Meiering Elizabeth M EM Kay Lewis E LE

Proceedings of the National Academy of Sciences of the United States of America 20161024 45

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD1<sup>2SH</sup>, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD1<sup> ...[more]

PMID: 27791136

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Project description:Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein-protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid β-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Dataset Information

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Publications

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

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