**2. Structure and function of Cu, Zn-superoxide dismutase (SOD1)**

Before discussing SOD1 folding, misfolding and aggregation, we will give a brief description of the tertiary and quaternary structure of SOD1. Human SOD1 is a 32 kDa homodimeric metalloenzyme, with each subunit consisting of a 153 amino acid chain that is often N-terminally acetylated, contains a highly conserved, intrasubunit disulfide bond, and binds one Cu ion and one Zn ion (Figure 2A, C).

Each monomer folds into a Greek key β-barrel, comprised of two, four-stranded antiparallel β-sheets arranged at an angle with respect to one another. The β-barrel has a noncontinuous topology such that strands 1 through 3 together with strand 6 form the first βsheet, while strands 4, 5, 7 and 8 form the second β-sheet. The β-strands are connected by seven loops that differ greatly in length. Loop IV and loop VII are known as the metal binding and electrostatic loops, respectively, and play important roles in both stability and catalytic function by binding the metals and forming the catalytic site pocket. In addition to forming the Zn-binding site, Loop IV contains residues that are important for dimer interface and intrasubunit disulfide bond formation, which tethers Loop IV to β-strand 8 (Tainer et al. 1982). Thus, Zn binding, disulfide bond formation and dimerization together stabilize the native conformation of this long loop, greatly affecting the overall stability of the protein. Loop VII mainly plays a functional role, containing charged residues that shield the active site. These charged residues are important for guiding the superoxide anion from the surface of the protein into the active site where the redox active Cu ion is bound (Valentine et al. 2005).

SOD1 is abundant and ubiquitously expressed in the cytosol of aerobic organisms (Brown et al. 2004). Maturation of the protein involves a series of post-translational modifications, which are understood to varying extents. When it is initially synthesized in the reducing environment of the cytosol the protein is thought to adopt a marginally stable, folded, monomer structure with a reduced disulfide bond and no bound metals. How the protein acquires Zn is not known; however, Cu can be acquired by interaction with the copper chaperone for SOD1 (CCS) or by a CCS-independent mechanism that may involve glutathione, but that is not well understood (Leitch et al. 2009). CCS also catalyzes intrasubunit disulfide bond formation in the reducing cellular environment (Leitch et al. 2009). Although the most abundant form of SOD1 is usually the native, fully mature,

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 273

A common and powerful approach to understanding the molecular basis for aggregate formation is to investigate the biophysical properties of mutant proteins in the native and unfolded states, as well as any equilibrium or kinetic intermediates that arise as the protein folds or unfolds (Dobson 2004). Equilibrium species refer to the most stable conformations that are significantly populated under specific steady-state conditions, while kinetic species refer to conformations that are transiently populated as an unfolded protein folds into its native conformation. Typically, kinetic folding intermediates have a relatively low energy barrier of formation, and therefore can form quickly, but they are generally not the most stable conformations. Before protein folding has reached equilibrium (ie. during kinetic conditions), it is not the stability of each state that determines the relative population of each species along the folding/unfolding pathway, but rather how rapidly these states can be accessed on the time scale of protein folding/unfolding. Investigating the molecular characteristics that govern the stability of different states and enable efficient folding of SOD1 can provide key insights into the cause of ALS (Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006; Rumfeldt et

In recent years, systematic analyses of the effects of fALS-associated mutations on the stability and folding of various forms of SOD1, including holoS-S, apoS-S and apoSH, have been reported. Human SOD1 contains two free cysteine residues at amino acid positions 6 and 111 (Figure 2), and these free cysteine residues inhibit reversible unfolding of SOD1 *in vitro* by forming intramolecular and intermolecular non-native disulfide bonds, which promote SOD1 aggregation (Lepock et al. 1990; McRee et al. 1990). Reversible unfolding is a prerequisite for thermodynamic analysis, and so to overcome this limitation pseudo-wild type (pWT) constructs lacking these free cysteines have been used extensively for *in vitro* studies of SOD1. In the most widely used pWT construct, the free cysteines are mutated to alanine and serine at positions 6 and 111, respectively (Lepock et al. 1990; McRee et al. 1990; Stathopulos et al. 2003; Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006; Kayatekin et al. 2008; Rumfeldt et al. 2009; Vassall et al. 2011); however, other mutations at these positions have also been used (most notably C6A and C111A) (Lindberg et al. 2004; Nordlund and Oliveberg 2006; Nordlund et al. 2009). Not only are these chemically and structurally conservative mutations, a serine at position 111 is found in most other mammalian SOD1, and alanine at position 6 is observed in other non-mammalian organisms (Getzoff et al. 1989). Mutating the free cysteines results in highly reversible unfolding of pWT, while having very minimal effects on structure, function and stability (Lepock et al. 1990; McRee et al. 1990; Hallewell et al. 1991; Parge et al. 1992; Vassall et al. 2011). In addition, an engineered monomer construct (pWT*mon* SOD1) has been used to investigate the effects of ALS mutations on the stability and folding behaviour of individual SOD1 subunits (Nordlund and Oliveberg 2006; Hornberg et al. 2007; Kayatekin et al. 2008; Nordlund et al. 2009; Kayatekin et al. 2010). The monomer construct contains two glutamic acid residues in place of Phe50 and Gly51, and the presence of these charged residues in the dimer interface prevents SOD1 dimerization (Bertini et al. 1994; Banci et al. 1998). The use of both pWT and pWT*mon* SOD1 constructs has provided valuable insights into the mechanism of SOD1 folding and misfolding, which are described in the following sections, starting with the most

**3. Folding, unfolding and misfolding of SOD1** 

al. 2009; Vassall et al. 2011).

immature to most mature form of SOD1.

dimeric protein (Valentine et al. 2005), there is also evidence for a significant pool of SOD1 that lacks bound Cu, and is activated in response to oxidative stress (Brown et al. 2004). From here we will refer to the various states of the protein in terms of disulfide and metallation status, with a focus on the disulfide reduced (2SH), disulfide oxidized (S-S), fully metallated (holo) and metal free (apo) states.

The crystal structure of fully mature SOD1 is shown in panel A (pdb: 1HL5) (Strange et al. 2003). The β-strands are shown in gray and labelled in white starting from the N-terminus; the functional loops IV and VII are shown in red and blue, respectively; the conserved disulfide bond (S-S) between Cys57 and Cys146 is indicated by orange spheres, the nonconserved free Cys (6 and 111) are shown in cyan; and Cu and Zn are indicated by yellow and green spheres, respectively. The solution structure of the metal free SOD1 monomer variant (pWT*mon*, refer to the introduction of section 3) is shown in panel B (pdb: 1RK7) (Banci et al. 2003). In this structure, only β-strands 1-3 and 6 are well defined. The colour scheme in panel B is identical to panel A. The primary sequence of the SOD1 monomer is shown in panel C. The colours used to depict the secondary structure elements are identical to those used in panels A and B. The positions of known fALS-associated mutations are listed vertically in red below the naturally occurring amino acid in black. The yellow and green spheres indicate the metal binding residues and the secondary structure elements are listed above the primary sequence.

dimeric protein (Valentine et al. 2005), there is also evidence for a significant pool of SOD1 that lacks bound Cu, and is activated in response to oxidative stress (Brown et al. 2004). From here we will refer to the various states of the protein in terms of disulfide and metallation status, with a focus on the disulfide reduced (2SH), disulfide oxidized (S-S),

The crystal structure of fully mature SOD1 is shown in panel A (pdb: 1HL5) (Strange et al. 2003). The β-strands are shown in gray and labelled in white starting from the N-terminus; the functional loops IV and VII are shown in red and blue, respectively; the conserved disulfide bond (S-S) between Cys57 and Cys146 is indicated by orange spheres, the nonconserved free Cys (6 and 111) are shown in cyan; and Cu and Zn are indicated by yellow and green spheres, respectively. The solution structure of the metal free SOD1 monomer variant (pWT*mon*, refer to the introduction of section 3) is shown in panel B (pdb: 1RK7) (Banci et al. 2003). In this structure, only β-strands 1-3 and 6 are well defined. The colour scheme in panel B is identical to panel A. The primary sequence of the SOD1 monomer is shown in panel C. The colours used to depict the secondary structure elements are identical to those used in panels A and B. The positions of known fALS-associated mutations are listed vertically in red below the naturally occurring amino acid in black. The yellow and green spheres indicate the metal binding residues and the secondary structure elements are

fully metallated (holo) and metal free (apo) states.

Fig. 2. The structural elements of SOD1.

listed above the primary sequence.
