For superoxide dismutase 1 (SOD1), the metalloenzyme implicated in some forms of familial amyotrophic lateral sclerosis (ALS), the very features that allow the protein to do its job may make it prone to form abnormal aggregates. So conclude the authors of a structural biology study published online in PNAS this week. In a tour de force encompassing folding assays, NMR, calorimetry, and protein crystallography, the scientists in the laboratory of Mikael Oliveberg at Stockholm University in Sweden analyzed the properties of SOD1 when they took away the residues that normally bind a zinc ion. They determined that the zinc is present to stabilize the molecule’s otherwise floppy catalytic loops.

To mature, SOD1 acquires copper and zinc cofactors, forms intramolecular disulfide bonds, and dimerizes. Other studies have pointed to a role for the metal ions in stabilizing SOD1 structure; for example, the Apo form of the protein is more likely to aggregate (see ARF related news story and Banci et al., 2009). In the current study, joint first authors Anna Nordlund and Lina Leinartaité and colleagues found that when they removed the zinc-binding site from the protein, it was more prone to aggregation. “There is probably now enough evidence to consider these findings irrefutable,” wrote Jeffrey Agar of Brandeis University in Waltham, Massachusetts, in an e-mail to ARF. Agar was not involved in the Stockholm study.

SOD1’s copper is functionally required for the enzyme to neutralize free radicals, but the zinc appears to play a more structural role, Oliveberg wrote to ARF. To isolate the importance of the zinc, the scientists knocked out its binding site, mutating the ion’s ligands H63, H71, H80, and D83 to serine. The mutant protein still folded like the wild-type—but then held its structure more stably than the normal protein. The mutant SOD1 was less likely to unfold, and able to withstand higher concentrations of urea, than the native protein. Thus, the residues needed to bind zinc in SOD1’s mature form actually make it less stable when the protein is still immature.

The mutant protein still picked up a zinc ion, but did so with the sites that usually bind copper, which couldn’t position it properly. X-ray crystallography and NMR showed that the misaligned metal ion forced the protein’s catalytic loops to swing outward in a disordered fashion. Those wildly swaying loops, then, were more likely to reach out and bind another SOD1 monomer, potentially seeding the aggregation process. In contrast, the same loops in metal-bound SOD1 were rigid. Oliveberg concluded that the zinc is present to keep those loops tied down.

Oliveberg suggested that SOD1’s inclination toward aggregation is the result of an evolutionary tug-of-war between enzyme function and protein stability. “Like the long neck of a giraffe can sometimes cause problems, the long functional loops of SOD1 compromise the protein’s structural integrity,” he wrote. “Misfolded intermediates accumulate because the functional parts of the protein pull in the wrong direction.” Studies show that a few other proteins, such as the trefoil proteins, have a similar folding dilemma—the scientific term is “frustrated” (Capraro et al., 2008). SOD1 is the first such protein to be implicated in disease, Oliveberg wrote.

SOD1 is a highly conserved, ancient protein, and may be a sort of “molecular dinosaur,” Oliveberg wrote. It may now be so specialized that any mutation throws the protein off balance. “Perhaps it has painted itself into an evolutionary cul-de-sac: the function is essential, but puts a lot of ‘strain’ on the structure, so mutations are difficult to tolerate.” That would help explain why more than 100 different mutations in SOD1 have been linked to inherited ALS.—Amber Dance.

Nordlund A, Leinartaité L, Saraboji K, Aisenbrey C, Gröbner G, Zetterström P, Danielsson J, Logan DT, Oliveberg M. Functional features cause misfolding of the ALS-provoking enzyme SOD1. PNAS Early Edition. Abstract


  1. In a study of epic proportions (X-ray crystallography, calorimetry, NMR, melting, refolding, etc.), Oliveberg and coworkers add to the body of evidence showing that the loss of metals, and loss of zinc in particular, destabilizes SOD1 and promotes aggregation. There is probably now enough evidence to consider these findings irrefutable. They add considerably to the single prior study that demonstrates that the SOD1 electrostatic loop promotes protein aggregation. The authors use the frustrated model (for the non-scientist, this is a theory about how proteins fold, or don't, not Kate Moss at a buffet) to describe how the very residues that stabilize active SOD1 destabilize the demetallated enzyme. Oliveberg takes the loss of metals from wtSOD1, which results in destabilization and aggregation in vitro, as evidence that the more common, sporadic, cases are caused by loss of metals.

    So the loss of metals must be a really bad thing for ALS patients, and sporadic ALS patients must have aggregated WT SOD1? Not exactly. The major protein in sporadic patients' inclusions appears to be TDP43, and only occasionally SOD1. If you had to choose an ALS mutation, you would do better to take H46R, which doesn't bind Cu but has more than 10 years’ mean survival, than A4V, which binds metals well but has less than a year’s mean survival.

    What does this mean for ALS patients? Despite my nitpicking, it is clear that ALS-causing mutations of SOD1 (and probably TDP43) cause the protein to aggregate. Since small aggregates are quite toxic, and large aggregates choke off axons and kill neurons, we ought to be trying to stop aggregation. How many of the more than 50 past ALS clinical trials specifically targeted protein aggregation? My count is zero. How many are planned? My count is zero specific aggregation inhibitors, and one potential aggregation inhibitor. If you believe Oliveberg's data, as I do (we have data from 13 fALS variants that also indicate the electrostatic loop is perturbed), the SOD1 electrostatic loop may be a logical target. If not SOD1, we should target TDP43.

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News Citations

  1. No Metal, No Stability: Structure of Apo SOD1

Paper Citations

  1. . Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants. Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):6980-5. PubMed.
  2. . Backtracking on the folding landscape of the beta-trefoil protein interleukin-1beta?. Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):14844-8. PubMed.
  3. . Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9667-72. PubMed.

Further Reading


  1. . Metalation of the amyotrophic lateral sclerosis mutant glycine 37 to arginine superoxide dismutase (SOD1) apoprotein restores its structural and dynamical properties in solution to those of metalated wild-type SOD1. Biochemistry. 2007 Sep 4;46(35):9953-62. PubMed.
  2. . How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?. Trends Biochem Sci. 2007 Feb;32(2):78-85. PubMed.
  3. . Posttranslational modifications in Cu,Zn-superoxide dismutase and mutations associated with amyotrophic lateral sclerosis. Antioxid Redox Signal. 2006 May-Jun;8(5-6):847-67. PubMed.
  4. . Mutant SOD1 instability: implications for toxicity in amyotrophic lateral sclerosis. Neurodegener Dis. 2005;2(3-4):115-27. PubMed.
  5. . The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status. J Biol Chem. 2004 Nov 12;279(46):47998-8003. PubMed.
  6. . The structure of holo and metal-deficient wild-type human Cu, Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis. J Mol Biol. 2003 May 9;328(4):877-91. PubMed.
  7. . Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9667-72. PubMed.

Primary Papers

  1. . Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9667-72. PubMed.