Metal Ions and SOD1 Structure

First characterized in 1969 [83], SOD1 is a homodimeric molecule characterized by an eight-stranded Greek key ^-barrel fold in each subunit [84]. The two polypeptides are related by a two-fold axis of rotation at the dimeric interface (Figure 1A), which buries approximately 660 A2 of solvent accessible surface area per polypeptide and is formed by a series of reciprocal hydrogen bonding, hydrophobic, and water-mediated interactions [81]. The wild-type SOD1 holoenzyme is unusually stable, retaining substantial activity in 4% SDS and 8 M urea [85] and having a melting point of ~95 °C [86]. The dimeric quaternary structure is important for catalytic function, as demonstrated by an engineered SOD1 monomer that retains only 10% of normal SOD1 activity [87].

Each subunit contains a copper- and zinc-binding site. The metal binding arrangement is unique in that one ligand, His63, binds simultaneously to both copper and zinc metals when the enzyme is in the cupric form (Figure 3). This ligand arrangement is unique to SOD1 and His63 is termed the 'imidazo-late bridge' [84]. The copper(II) ion is further coordinated by three additional histidine residues at positions 46, 48, and 120 in a distorted square planar arrangement [84,88-90]. Structural studies have revealed a fifth ligand to Cu(II), a water molecule approximately 2.2-2.5A from copper [84,88-93]. The coordination geometry changes to a pseudotrigonal planar arrangement during the catalytic cycle when the imidazolate bridge is lost as the copper ion becomes reduced (Reaction 1). The breaking and reforming of the imidazolate bridge to the copper ion during the catalytic cycle has been verified both spectroscopically and structurally [91,93-97]. Figure 3 shows that the zinc ion is bound by one aspartate residue, Asp83, the bridging His63 and two other histidine residues, His71 and His80, in a tetrahedral coordination geometry. Zinc does not have catalytic function, but instead functions to structurally stabilize SOD1 [86,98].

Each ^-barrel of the dimer is flanked by two major loop elements termed the 'electrostatic' and 'zinc' loops (Figure 1A). The electrostatic loop contains charged residues Glu132, Glu133, and Lys136 essential for electrostatic guidance of the substrate into the active site channel [17,19,99-104]. The active site channel is formed on one side by residues from the electrostatic loop and on the other side by residues of the zinc loop. The channel opening is wide at the entrance, approximately 24 A across, and narrows to about 4 A immediately above the copper ion [84]. The narrowing of the channel allows access to small anionic molecules such as azide, cyanide, and fluoride [93,105]. One of the most critical

Gly 85

Gly 85

Parkinson Disease Structure
Figure 3. Schematic diagram of the copper- and zinc-binding sites in SOD1. The ligand to the copper and zinc ions are labeled. His63 is the 'primary bridge' or the 'bridging imidazolate'. Asp124 is the 'secondary bridge' (see text). (Image taken from [150] and reproduced by permission).

residues for enzymatic activity, Arg143, sits approximately 6 A away from the copper ion and functions to correctly position the substrate relative to the active site in what is termed the 'anion binding site' [106]. Mutation of this residue results in an approximate 90% decrease in enzymatic activity [107,108]. Thr137 is positioned at the bottom of the active site channel and together with Arg143, sterically excludes larger nonsubstrate anions from reacting with the copper ion. The zinc and electrostatic loop elements are connected by the 'secondary bridge' of Asp124 (the bridging imidazolate is the 'primary' bridge). Asp124 links the two loop elements and the metal binding sites by making hydrogen bonds simultaneously to the zinc ligand His71 and to the copper ligand His46 (Figures 3 and 4). Thus, disruption of one loop element through mutation directly affects the mobility and the conformation of the other.

The intrasubunit disulfide bond between residues C57 and C146 is a feature conserved among SOD1 proteins across many species [109]. As shown in Figure 1A, it is an important element of SOD1 stability and function where it helps to hold firmly the structural elements that make up the homodimeric interface. Recent evidence suggests that transport of yeast SOD1 into the intermembrane space of mitochondria requires the loss of metal ions and reduction

Figure 4. Negative design of human SOD1 and the locations of the wild-type-like (WTL) and metal binding region (MBR) fALS mutations. In the left subunit, the edge strands of the ^-sheets and the gain-of-interaction (GOI) interface are boxed. In the wild-type protein, the zinc loop projects from the plane of the paper toward the viewer, preventing SOD1-SOD1 protein-protein interactions from occurring at the edge strands. The copper and zinc ions are shown as light and dark gray spheres, respectively. In the right subunit, the positions of the MBR mutations are shown as black spheres and the WTL mutations (not shown for clarity) are scattered throughout the ^-barrel. Metal binding stabilizes the conformation of the zinc and electrostatic loop elements and therefore plays an intimate role in the negative design of the molecule (see text).

Figure 4. Negative design of human SOD1 and the locations of the wild-type-like (WTL) and metal binding region (MBR) fALS mutations. In the left subunit, the edge strands of the ^-sheets and the gain-of-interaction (GOI) interface are boxed. In the wild-type protein, the zinc loop projects from the plane of the paper toward the viewer, preventing SOD1-SOD1 protein-protein interactions from occurring at the edge strands. The copper and zinc ions are shown as light and dark gray spheres, respectively. In the right subunit, the positions of the MBR mutations are shown as black spheres and the WTL mutations (not shown for clarity) are scattered throughout the ^-barrel. Metal binding stabilizes the conformation of the zinc and electrostatic loop elements and therefore plays an intimate role in the negative design of the molecule (see text).

of the disulfide bond, and that these are prerequisites for monomerization and/or unfolding of SOD1 [110]. Functionally, the reduced disulfide bond has been suggested to be recognized by the copper chaperone for superoxide dismutase during copper transfer from CCS to SOD1 [111]. Notably, all forms of SOD1 isolated through standard purification methods have its intrasubunit disulfide bond intact, presumably due to their exposure to air during the purification process. However, given the highly reducing environment of the cytosol [112,113], the status of this disulfide bond in vivo is uncertain.

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