Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients 178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.