Structural Characterization of Zinc-deficient Human Superoxide Dismutase and Implications for ALS

Abstract

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.

Reactive Nitrogen Species in Motor Neuron Apoptosis

“…Biochemical and biophysical studies indicate that the formation of a dimer between Zn-deficient SOD and Cu,Zn SOD increases the stability of the Zn-deficient SOD monomer Roberts et al 2007), in agreement with the in vivo and in vitro studies showing that wild type SOD increases the stability and solubility of the mutant protein (Fukada et al 2001;Witan et al 2008;Witan et al 2009). Moreover, in spite of Cu,Zn SOD being a very stable enzyme, the half-life for exchange between this enzyme and Zn-deficient SOD at 37°C is surprisingly fast at 13-17 min when determined using differential mobility gel electrophoresis and 14 min by FRET (Roberts et al 2007;Sahawneh et al 2010). An important observation is that the reassociation of Cu,Zn SOD monomers is approximately 10,000 times slower than the reassociation of apoSOD monomers (Lindberg et al 2004;Svensson et al 2006), suggesting that the alterations of the dimer interface in the apoSOD are responsibly for the faster association of the monomers even though the stability of the dimer is diminished when compared with Cu,Zn SOD.…”

“…In addition, Zn-deficient SOD toxicity is prevented by peptides that inhibit nitration (Ye et al 2007;Sahawneh et al 2010), as is the increased toxicity after the addition of Cu,Zn SOD (Sahawneh et al 2010). Biochemical and biophysical studies indicate that the formation of a dimer between Zn-deficient SOD and Cu,Zn SOD increases the stability of the Zn-deficient SOD monomer Roberts et al 2007), in agreement with the in vivo and in vitro studies showing that wild type SOD increases the stability and solubility of the mutant protein (Fukada et al 2001;Witan et al 2008;Witan et al 2009). Moreover, in spite of Cu,Zn SOD being a very stable enzyme, the half-life for exchange between this enzyme and Zn-deficient SOD at 37°C is surprisingly fast at 13-17 min when determined using differential mobility gel electrophoresis and 14 min by FRET (Roberts et al 2007;Sahawneh et al 2010).…”

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

“…S4). Because of the protease resistance of WT and G93A SOD1, likely conferred via metal binding (45) and an intramolecular disulfide bond (32,44,46,47), the G85R variant, which is less protease resistant, was used. The m/z 487.8 (M r 973.6; residues 1-9, acetylated N terminus) is observed in both samples and is presented as a positive control, highlighting similar intensities of the peptide in the cross-linked and non-cross-linked samples.…”

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

“…H46R, G85R, D124V, and S134N) show a structured ␤-barrel core with disorder of loops IV and VII (3,22,54,77). The importance of zinc binding to order loops IV and VII has been suggested by molecular dynamics simulations (78) and isothermal titration calorimetry (63); furthermore, bound copper is insufficient to order the loops in the absence of zinc binding (79). The dimer interface region of loop IV is tethered to the ␤-barrel by the disulfide linkage between Cys-57 and Cys-146, and either zinc acquisition or disulfide oxidation can convert disulfide-reduced apoSOD1 monomers to a dimeric form (48,56).…”