Disulfide-reduced ALS variants of Cu, Zn superoxide dismutase exhibit increased populations of unfolded species

J Mol Biol. 2010 Apr 30;398(2):320-31. doi: 10.1016/j.jmb.2010.02.034. Epub 2010 Feb 23.

Abstract

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral pH and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to approximately 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amyotrophic Lateral Sclerosis / enzymology*
  • Amyotrophic Lateral Sclerosis / genetics
  • Crystallography, X-Ray
  • Disulfides / chemistry*
  • Enzyme Stability
  • Humans
  • Mutation
  • Oxidation-Reduction
  • Protein Folding
  • Protein Multimerization
  • Ribosomes / metabolism
  • Superoxide Dismutase / biosynthesis
  • Superoxide Dismutase / chemistry*
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase-1
  • Zinc / chemistry*

Substances

  • Disulfides
  • SOD1 protein, human
  • Superoxide Dismutase
  • Superoxide Dismutase-1
  • Zinc