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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: Structure. 2016 Sep 22;24(11):1898–1906. doi: 10.1016/j.str.2016.08.011

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-associated Mutation

James M Fay 1,2,4, Cheng Zhu 1,4, Elizabeth A Proctor 1,2,3, Yazhong Tao 1, Wenjun Cui 1, Hengming Ke 1, Nikolay V Dokholyan 1,2,3,5
PMCID: PMC5093072  NIHMSID: NIHMS813878  PMID: 27667694

SUMMARY

The majority of amyotrophic lateral sclerosis (ALS)-related mutations in the enzyme Cu, Zn superoxide dismutase (SOD1), as well as a post-translational modification, glutathionylation, destabilize the protein and lead to a misfolded oligomer that is toxic to motor neurons. The biophysical role of another physiological SOD1 modification, T2-phosphorylation, has remained a mystery. Here, we find that a phosphomimetic mutation, T2D, thermodynamically stabilizes SOD1 even in the context of a strongly SOD1-destabilizing mutation, A4V, one of the most prevalent and aggressive ALS-associated mutations in North America. This stabilization protects against formation of toxic SOD oligomers and positively impacts motor neuron survival in cellular assays. We solve the crystal structure of T2D-SOD1 and explain its stabilization effect using DMD simulations. These findings imply that T2-phosphorylation may be a plausible innate cellular protection response against SOD1-induced cytotoxicity, and stabilizing the SOD1 native conformation might offer us viable pharmaceutical strategies against currently incurable ALS.

Graphical Abstract

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INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by gradual loss of motor neurons in the brain and spinal cord (Boillee et al., 2006; Cleveland and Rothstein, 2001; Redler and Dokholyan, 2012). Currently no effective therapeutics for ALS exist (Abel et al., 2012; Ilieva et al., 2009; Israelson et al., 2015). Genomic studies have associated approximately 12% of familial ALS cases and 1.5% of sporadic cases with mutations in the ubiquitous antioxidant enzyme Cu, Zn superoxide dismutase (SOD1) (Cirulli et al., 2015; Rosen et al., 1993). SOD1 aggregates have been discovered in the ventral horns of spinal cords from patients with familial or sporadic cases of ALS, indicating a contribution of non-genetic factors to disease progression (Shibata et al., 1996; Shibata et al., 1994). Recent studies have further implicated non-native SOD1 oligomers, instead of large aggregates, as the toxic species in cellular assays (Jonsson et al., 2004; Luchinat et al., 2014; Proctor et al., 2016; Zetterstrom et al., 2007). Indeed, several disease mutants (e.g. A4V and G93A) destabilize SOD1 and promote formation of a trimeric species (Proctor et al., 2016; Redler et al., 2014). Aberrant post-translational modifications such as glutathionylation, a result of oxidative stress (Barber et al., 2006; Townsend, 2007), also increase the rate of SOD1 oligomerization as well as the exposure of a toxicity-associated epitope (Bosco et al., 2010; Redler et al., 2014; Redler et al., 2011). Hence, understanding how somatic mutations and non-genetic factors affect the stability of the SOD1 dimer and non-native oligomers is vital to the development of novel therapeutic strategies. Identifying mechanisms to stabilize the native conformation of SOD1 and to eliminate potentially toxic species can shed light on new venues for drug discovery.

The stability of the SOD1 dimer is affected by ALS-related mutations and several post-translational modifications, which typically bias the SOD1 misfolding pathway toward dimmer dissociation and lead to larger populations of oligomers and aggregates (Broom et al., 2016; Khare et al., 2004; Redler et al., 2014). In familial and sporadic ALS cases, post-translational modifications induced by environmental factors play a significant role in disease progression (Barber and Shaw, 2010; Chattopadhyay et al., 2015; Pasinelli and Brown, 2006). Specifically, glutathionylation of C111, oxidative modification of W32, and phosphorylation of T2 or S58/T59 have been identified in human SOD1 (Coelho et al., 2014; Wilcox et al., 2009). While C111-glutathionylation and W32-oxidation have been studied thoroughly and linked to increased concentrations of non-native oligomers (Coelho et al., 2014; Redler et al., 2011), studies on phosphorylation have been limited due to a lack of knowledge of the corresponding kinases. A recent study demonstrated that S59-phosphorylated SOD1 relocates to the nucleus and functions as a transcription factor regulating oxidative resistance genes (Tsang et al., 2014). However, the effects of T2-phosphorylation, especially its impact on protein stability, have remained elusive.

Experimental characterization of phosphorylated SOD1 has been limited due to the inability to isolate pure T2-phosphorylated SOD1 or to artificially produce it via kinase reaction (Goscin and Fridovich, 1972). Phosphorylated SOD1 can be enriched through ion exchange chromatography, but the resulting sample is impure, containing a large proportion of unmodified and alternatively phosphorylated SOD1 (T2 or S58/T59) (Wilcox et al., 2009). Here we address this problem by producing the phosphomimetic mutant T2D-SOD1. We explore the properties of phosphomimetic mutant using a synergistic computational and experimental approach. Using molecular dynamics simulations, we demonstrate similarities between T2D-SOD1 and T2-phosphorylated SOD1 in terms of their energetic, thermodynamic, and structural characteristics, thus supporting the use of T2D as a phosphomimetic mutation. We solve the crystal structure of T2D-SOD1, and find that it closely matches the wild type (WT-SOD1) structure (Deng et al., 1993). In agreement with our computational predictions, the phosphomimetic T2D mutation stabilizes the SOD1 dimer without altering its native structure and preserves the same level of cellular viability when compared to WT-SOD1. In order to assess the role of phosphorylation in disease, we test the ability of the T2D mutation to mitigate the effects of an aggressive ALS mutation A4V. By expressing T2D-SOD1, A4V–SOD1 and T2D/A4V–SOD1 in hybridized motor neuron cells, we find that the T2D mutation rescues the cytotoxic effect of A4V–SOD1, demonstrating the benefit of stabilizing the SOD1 native conformation and offering a plausible pharmaceutical strategy to fight ALS.

RESULTS

Computational analysis suggests common characteristics shared by T2-phosphorylated SOD1 and T2D-SOD1

We perform all-atom DMD simulations to test whether the physical properties of the phosphomimetic mutant T2D-SOD1 reproduce those of T2-phosphorylated SOD1 (Pi-SOD1). We compare energetic, structural, and thermodynamic signatures of T2D-SOD1 and Pi-SOD1 (Figure S1). Specifically, we compare the potential energy distribution of each species, which are obtained through replica exchange simulations. The free energy histogram contains three distinct peaks representing three co-existing metastable states: a native low-energy dimeric structure, a partially unfolded dimer, and dissociated monomers with partial unfolding. T2D-SOD1 and Pi-SOD1 both have enriched populations of native dimers (peak 1) and decreased populations of dissociated monomers (peak 3) (Figure S1A, B), indicating reduced amounts of nonnative states in both species in comparison with the unmodified SOD1 (WT-SOD1). We also compare melting temperatures (Tm) corresponding to the unfolding of SOD1 monomers through specific heat analysis (Ding and Dokholyan, 2008). We find that the Tm of both T2D-SOD1 and Pi-SOD1 are lower than that of WT-SOD1 (Figure S1C, D). Furthermore, Pi-SOD1 exhibits a major peak with a shoulder in the specific heat plot against temperature (Figure S1C, D), in contrast to WT-SOD1 that shows a single peak in heat capacity. The single peak suggests the dimer dissociation and monomer unfolding are coupled processes for unmodified SOD1 and the shoulder peak represents a partial decoupling induced by phosphorylation (Proctor et al., 2011). We find that T2D-SOD1 reproduces this feature by preserving this decoupling. Together these results reveal similar monomer and dimer stabilities for T2D-SOD1 and Pi-SOD1.

The T2D-SOD1 dimer is structurally similar to the WT-SOD1 dimer

In order to study the structural effect of the phosphomimetic mutation T2D, we solve the crystal structure of T2D-SOD1 to resolution of 2.2 Å (PDB: 5K02, Table 1). Structure solution by molecular replacement with the model of WT-SOD1 (PDB: 1SPD) reveals a dimeric β-barrel occupying the center of an asymmetric unit (Figure 1). In the T2D-SOD1 crystals, the packing between the central dimers leads to a ternary complex in space group P1 (Figure S2). The structures of T2D-SOD1 and WT-SOD1 are similar to each other. By superimposing their backbone and side chains, we find that T2D-SOD1 deviates from WT-SOD1 with root mean square deviation of 0.93 Å (Figure 1A). This deviation largely originates from the flexible loops near the N terminus (Figure 1B). CD spectra also indicate that T2D-SOD1 and WT-SOD1 have similar secondary structure composition in solution (Figure 1C). To quantitate the rearrangement induced by the T2D mutation, we calculate the rotation angles between two monomers using previously described algorithms (Proctor et al., 2011). For T2D-SOD1 and WT-SOD1 these angles are 36.0° and 33.6°, respectively. Our DMD simulation predicts that both the T2D mutation and T2-phosphorylation induces an increase in the average monomer-monomer angle (Figure S3), suggesting that the T2D mutation may structurally mimic the steric effects of SOD1 phosphorylation at T2. Overall the crystal structure confirms that no large rearrangement of subunits or disruption of the dimer interface occurs in T2D-SOD1.

Table 1. Data collection and refinement statistics.

Data Collection
Space Group P1
Cell Dimensions
    a,b,c (Å) 111.980, 111.961, 150.46
    α,β,γ (°) 89.86, 89.85, 60.13
Resolution (Å) 40.00–2.10 (2.14–2.10)
Rmerge (%) 6.1
I / σI 18.3 (1.6)
Completeness (%) 80.41
Redundancy 2.5 (2.3)
Refinement Statistics
Resolution (Å) 40.00–1.99
No. Reflections 329360
Rwork / Rfree (%) 14.6/16.2
No. atoms
    Protein Atoms 27396
    Ligand/ion 48
    Water 686
Overall B value (Å2) 36.11
    Protein 36.01
    Ligand/ion 34.50
    Water 40.09
RMSD
    Bond Lengths (Å) .006
    Bond Angles (°) 1.084
Ramachandran
    Most Favored (%) 87.0
    Additionally Allowed (%) 12.9
    Generously Allowed (%) 0.1
    Disallowed (%) 0.0
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Figure 1. T2D-SOD1 and WT-SOD1 have similar structures in crystals and in solution state.

Figure 1

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(A) Overall ribbon diagram of the T2D-SOD1 homodimer (PDB: 5K02) with Zn2+ (silver) and Cu2+ (golden) shown in spheres. The superposition of T2D- (light green and light brown) and WT-SOD1 (purple, PDB: 1SPD) in one subunit indicates the minor structural change in the loop regions. The side chains of D2 and A4 are indicated as cyan sticks. (B) Electron density map of D2 residue and surrounding residues (i. 22–29, 107–113) in one subunit. (C) CD spectra of T2D-SOD1 (cyan square) and WT-SOD1 (orange circle) indicate they both have β-sheet structure in solution. See also Figure S1, S2 and S3.

T2D mutation promotes the native dimer population in both WT- and A4V–SOD1

Many disease-linked SOD1 mutants undergo dimer dissociation with greater frequency than WT-SOD1, leading to an increase in the populations of monomeric species that form the available pool for further oligomerization (Broom et al., 2015; Khare and Dokholyan, 2006). In contrast, we found that T2D-SOD1 maintained a stable dimer population after incubation at 37 °C for up to seven days (Figure 2A). For comparison, WT-SOD1 forms a small population of monomeric proteins during the same time period (Figure 2A). Glutathionylation at C111 has been shown to promote SOD1 dimer dissociation in both wild type and mutant SOD1 (e.g. A4V–SOD1) by inducing steric clashing near the dimer interface (Redler et al., 2011; Proctor et al., 2011). The small difference in the monomer-dimer population distribution between T2D- and WT-SOD1 is exacerbated by glutathionylation. As seen previously, glutathionylated WT-SOD1 (GS-WT-SOD1) features an increased population of monomer as compared to unmodified WT-SOD1, yielding up to 50% of the entire protein population after an incubation period of seven days at 37°C (Figure 2B). However, GS-T2D-SOD1 and GS-T2D/A4V–SOD1 resist dimer dissociation, featuring significantly smaller proportions of monomer than GS-WT-SOD1. Furthermore, A4V–SOD1 has previously been shown to have a higher propensity to form both monomer and oligomers (Luchinat et al., 2014; Redler et al., 2014). However, no formation of higher oligomer is evident in any of the phosphomimetic samples (Figure 2 and Figure S4). These results suggest that the phosphomimetic T2D mutation prevents or decreases SOD1 dimer dissociation.

Figure 2. T2D stabilizes the native state of SOD1 through decreased dimer dissociation rates.

Figure 2

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(A) Size exclusion chromatograms showing the populations of native dimer (15.5 ml) and monomer (17.4 ml) for unmodified T2D-, T2D/A4V- and WT-SOD1. Samples were taken after incubation at physiological conditions (30 µM SOD1, pH 7.4) and 37°C for 7 days. (B) Size exclusion chromatograms for glutathionylated T2D-, T2D/A4V- and WT-SOD1. Dissociation of immobilized dimers was monitored by surface plasmon resonance for T2D-, T2D/A4V- and WT-SOD1 (C) and glutathionylated species (D). Thermal denaturation curves of unmodified (E) and glutathionylated(F) SOD1 proteins indicate the Tm of T2D-SOD1 is lower than that of WT-SOD1 in the unfolding of SOD1 monomers. Fitting to a two-state model is represented as lines. Size exclusion chromatograms, dissociation profile and thermal denaturation curves of A4V–SOD1 and GS-A4V–SOD1 have been reported (Redler et al., 2011). See also Figure S4.

T2D mutation strengthens interactions at the SOD1 dimer Interface

In order to explore the mechanism of T2D dimer stabilization, we measure the dimer dissociation rates using SPR. In the case of glutathionylation-enriched SOD1 samples, Redler, et al showed that reassociation of previously dissociated monomers is inhibited (Redler et al., 2011). Together these studies determined the dissociation rate constants for WT-SOD1 and A4V–SOD1 as 1.72×10−4 s−1 and 2.60 ×10−3 s−1, respectively, indicating that A4V–SOD1 dissociates 15 times faster than does the WT-SOD1. We measure the dissociation rate constants for T2D-SOD1 as 1.01×10−4 s−1, approximately half that of WT-SOD1 (Table 2 and Figure 2C). For the glutathionylation-enriched samples, the rate constant of GS-T2D-SOD1 is reduced by a factor of 9 compared to GS-WT-SOD1, indicating GS-T2D-SOD1 dissociated significantly slower with an average half-time of 1.85 hr, in comparison to a half-time of 0.29 hr for GS-WT-SOD1 (Table 2 and Figure 2D). The dissociation rates of unmodified and glutathionylated T2D/A4V–SOD1 are similar to the respective values for T2D-SOD1 (16 times slower than A4V–SOD1), indicating an overall decrease in dimer dissociation consistent with our results from size exclusion chromatography. Because dimer dissociation and subsequent metal loss induce the structural distortions that promote protein aggregation, the decrease we find in dimer dissociation upon phosphomimetic T2D mutation suggests that phosphorylation at T2 may inhibit the formation of toxic SOD1 oligomers (Proctor et al., 2016).

Table 2. Biophysical characterization of T2D-, T2D/A4V- and WT-SOD1.

T2D, T2D/A4V and WT indicate unmodified species, GS-T2D, GS-T2D/A4V and GS-WT indicate C111-glutathionylation enriched species. Thermal melting temperatures (Tm) for the unfolding process of SOD1 monomers were measured by circular dichroism thermal denaturation. Dimer dissociation rate constants (koff) were measured by surface plasmon resonance. The standard deviation is indicated with ±.

Tm (°C) koff (s−1)
T2D 79.8 ± 1.5 1.01×10−4 ± 5.0×10−6
T2D/A4V 82.4 ± 0.7 1.56×10−4 ± 0.1×10−6
WT 81.5 ± 0.4 1.72×10−4 ± 0.2×10−6
GS-T2D 77.0 ± 0.5 1.50×10−4 ± 2.8×10−5
GS-T2D/A4V 77.0 ±0.4 1.61×10−4 ± 0.2×10−6
GS-WT 81.0 ± 1.8 9.49×10−4 ± 1.2×10−5
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T2D mutation rescues cell viability in the context of an FALS mutation A4V

In order to gain an understanding of the physiological relevance of the phosphomimetic mutant SOD1 in cellular environment, we overexpress T2D-SOD1 and WT-SOD1 in immortalized mouse motor neuron-like cells (NSC-34) with similar expression levels for wild type and mutants (Figure 3) (Cashman et al., 1992). We measure the cell viability using propidium iodide to mark non-viable cells, in concert with Hoescht staining of viable cells (Figure 3A) (Latt and Stetten, 1976; Moore et al., 1998). Overexpression of T2D-SOD1 and WT-SOD1 yield cell death rates of 7% and 6%, respectively (Figure 3B), which are identical results within experimental error. For a disease-relevant comparison, we similarly overexpress an aggressive ALS mutant A4V–SOD1 and the double mutant T2D/A4V–SOD1 in NSC-34 cells. While A4V–SOD1 expression results in 20% cell death, T2D/A4V–SOD1 expression results in only 6% cell death, demonstrating the ability of the T2D mutation to rescue toxicity of one of the most aggressive ALS-associated SOD1 variants. Supporting this finding, we find that cells expressing SOD1 with the A4V mutation alone feature high expression of the apoptotic marker caspase3, in agreement with the known toxicity of the A4V–SOD1 mutant, while cells transfected with T2D/A4V–SOD1 express lower levels of caspase3, similar to the levels expressed by cells transfected with WT-SOD1 (Figure 3C). These results indicate that the T2D mutation ameliorates cytotoxicity in the context of a severe ALS related mutation.

Figure 3. T2D mutation reduces the cytotoxic effect of A4V in motor neuron-like cells.

Figure 3

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(A) Transfection of NSC-34 cells with T2D-SOD1 and WT-SOD1 (negative control) led to similar cell death ratio. A4V–SOD1 transfection (positive control) decreased cell viability and T2D/A4V–SOD1 rescued cells from A4V–SOD1 mediated cytotoxicity. Applied three days post-transfection, red stain (propidium iodide) identifies dead cells, while blue stain (Hoechst) identifies all cell population. (B) Average cell death rates are measured as the percentage of red stained cells: WT 5.4%, T2D 5.6%, A4V 20.7% and T2D/A4V 6.0%. Each bar represents the standard error of the mean, n=3. (C) Levels of the apoptotic marker cCaspase 3 in NSC-34 cells demonstrated by western blot confirm the protection role of T2D mutation, as T2D/A4V leads to reduced expression of cCaspase 3. The normalized expression levels: WT 100%, T2D 89%, A4V 377% and T2D/A4V 135%.

Possible destabilizing effect of the T2D mutation on SOD1 trimers

Since cytotoxicity in motor neuron-like cells is found to correlate with the propensity of forming trimeric SOD1 (Proctor et al., 2016), we study the effect of T2D mutation on the stability of toxic SOD1 trimers. We use structural models of the trimer proposed by Proctor et al (Proctor et al., 2016) to computationally evaluate the change in free energy upon mutation (ΔΔGmut) using the Eris molecular design suite (Yin et al., 2007a, b). The T2D mutation destabilizes the SOD1 trimer structure (ΔΔGmut = 2.9 kcal/mol) (Figure S5), while the A4V mutation stabilizes the trimer (ΔΔGmut = −4.8 kcal/mol), in accordance with experimental observations (Redler et al., 2014). When combined in the same structure, the T2D mutation rescues the trimer-stabilizing effect of the A4V mutation (ΔΔGmut = 1.2 kcal/mol), suggesting that not only is the T2D mutation stabilizing to the native SOD1 dimer structure, but also inhibits formation of the toxic trimeric form by making the trimer structure less stable. Therefore, the phosphomimetic T2D mutation has a profound effect on SOD1 stability: by both stabilizing native dimer and destabilizing the toxic trimer states, this mutation strongly inhibits formation of toxic oligomers and promotes significant increase in cell viability.

Previous studies indicate that ALS-associated mutations and aberrant post-translational modifications can disrupt the dynamic coupling between SOD1 monomers, which leads to disruption of stabilizing structural elements (the β-plug and zinc-binding loop) and global destabilization of the native state (Ding and Dokholyan, 2008; Khare and Dokholyan, 2006). To understand whether and how phosphomimetic mutation affect the dynamics of SOD1 dimer on a nanosecond time scale, we have performed short (50 ns) DMD simulations and observed different dynamic signatures of WT-SOD1, T2D-SOD1, T2D/A4V–SOD1, and A4V–SOD1 (Figure 4 and Figure S6). In contrast to WT, T2D, and T2D/A4V, the A4V mutation leads to higher fluctuation of the residues at dimer interface, as measured by root mean square fluctuation in the DMD trajectories (Figure 4). Specifically, V148-V148 and F50-I151 are key hydrophobic contacts between the SOD1 monomers. We observe high flexibility of Val148 and Ile 151 only in the A4V–SOD1 (Figure 4B, green dash line). We also find that T2/A4 and V148/151 have cross-correlated motions in the inter-residue covariance matrix (Figure S6), which indicates that the packing strain induced by the T2D or A4V mutation can affect these residues. The A4V mutation likely induces steric clash at the interface or weakens the V148-V148 and F50-I151 interactions, whereas in T2D-SOD1 and T2D/A4V–SOD1 these interactions are stable (Figure 4C, D). This observation is consistent with the high propensity of A4V–SOD1 to dissociate and form monomers in comparison with WT-SOD1, T2D-SOD1 and T2D/A4V–SOD1. Furthermore, we find strong correlations within the zinc-binding loop (residues 49–60) only in A4V–SOD1, whereas in WT-, T2D-, and T2D/A4V–SOD1 these correlated motions are suppressed (Figure S6). The zinc-binding loop has been suggested to allosterically regulate the conformation of the C4F6 epitope (Bosco et al., 2010), which is directly linked to neural-toxicity of SOD1 in a primary microglia activation assay (Furukawa et al., 2015; Rotunno et al., 2014). We propose that in A4V–SOD1 the cross-correlated motions promote the exposure of the disease-related epitope, and that the T2D/A4V double mutant prevents this effect. These effects may lead to the differential cytotoxicity observed in cells transfected with A4V–SOD1 and T2D/A4V–SOD1.

Figure 4. DMD simulation suggests the key interface interactions are disrupted in A4V–SOD1, in contrast to WT-, T2D- and T2D/A4V–SOD1.

Figure 4

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The flexibility of the protein backbone is measured as root mean square fluctuation (RMSF, Red: 3.5 Å, Blue: 0.5 Å) and the results are an average of ten DMD trajectories: (A) WT-SOD1, (B) A4V–SOD1, (C) T2D-SOD1 and (D) T2D/A4V–SOD1. The hydrophobic contact between V148-V148 is indicated as green dash line. The zinc-binding loop (Zn-loop) which allosterically modulates the C4F6 epitope is indicated in the WT-SOD1 structure. See also Figure S5 and S6.

DISCUSSION

Recent studies suggest that non-native SOD1 oligomers are the cytotoxic species that promote disease progression and cause motor neuron degeneration in SOD1-mediated ALS (Jonsson et al., 2004; Proctor et al., 2016; Zetterstrom et al., 2007). The formation of SOD1 oligomers and aggregates is critically dependent on the dissociation of the native dimer into monomers, a process exacerbated by disease-linked mutations (Ding et al., 2012; Ivanova et al., 2014; Khare et al., 2004b). By reducing the population of SOD1 monomers, the formation of cytotoxic oligomers can be inhibited. Our findings demonstrate that the T2D mutation, which mimics phosphorylation at T2, increases the stability of the native conformation of SOD1 and reduces the cytotoxic effect of A4V–SOD1 in motor neuron-like cells. Furthermore, we suggest that T2-phosphorylation may be an intrinsic protective mechanism that stabilizes cytosolic SOD1. Our simulations and structural calculations for T2D-SOD1 and Pi-SOD1 not only demonstrate the stabilizing effect of the T2D mutation, but also establish the high degree of structural and energetic similarity between the phosphomimetic T2D-SOD1 construct and phosphorylated WT-SOD1. Quantitative analysis of the crystal structure of T2D reveals that T2D-SOD1 has a larger monomer-monomer angle in comparison to WT-SOD1, as predicted for the Pi-SOD1 (Figure S4). We also measure the thermal melting temperature (Tm) for T2D-SOD1 and find that T2D-SOD1 has a lower Tm than WT-SOD1 (T2D-SOD1: 79.8°C, WT-SOD1: 81.5°C) (Table 2 and Figure 2 E , F), which agrees with the shift of corresponding specific heat (Cv) peak to a lower temperature for Pi-SOD1 (Figure S1C, D). Based on these results, we posit that T2-phosphorylation of SOD1 has similar biophysical characteristics to T2D-SOD1 and stabilizes the native conformation of SOD1 dimer.

T2-phosphorylation and C111-glutathionylation have been identified as the dominant post-translational modifications to SOD1 in human erythrocytes (Wilcox et al., 2009). It is plausible that the phosphorylation is an intrinsic mechanism to offset the impact of oxidative stress or other deleterious effects on the SOD1 structure. The late onset of ALS symptoms (∼55 years old on average) suggests that such mechanisms may have evolved to protect the native SOD1 dimer (Ilieva et al., 2009; Redler and Dokholyan, 2012). Given the high cytosolic concentration (30–100 µM) of SOD1, a balancing mechanism that would offset environmental stressors, such as oxidative stress, may be important for the stability and function of SOD1 (Jaarsma et al., 2000). The protective role of phosphorylation has been discovered in other neurodegenerative diseases. In a rat model of Parkinson`s disease, researchers have applied phosphomimetic mutations and suggested phosphorylation at S87 inhibits the aggregation of human α-synuclein and protects against its toxicity (Oueslati et al., 2012). However, the roles of phosphorylation in neurodegenerative diseases are usually deleterious. Phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis (Rezaei-Ghaleh et al., 2016), reduce binding of Tau-protein to microtubules and correlate with severity of neuronal cytopathology (Augustinack et al., 2002; Lindwall and Cole, 1984). Together our findings and other studies reveal that while C111-glutathionylation promotes dimer dissociation by inhibiting the association of SOD1 monomers (decreased kon) (McAlary et al., 2013; Redler et al., 2011), T2-phosphorylation may inhibit dimer dissociation by stabilizing the dimer interface (decreased koff) (Figure 5).

Figure 5. A proposed mechanism of the regulation of dimer-monomer equilibrium in SOD1 by post-translational modifications.

Figure 5

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T2-Phosphorylation and C-111 glutathionylation may regulate the dimer-monomer equilibrium and have opposing effects on the stability of native SOD1 dimers. The T2D mutation (or T2-Phosphorylation) inhibits the dimer dissociation (decreased koff), whereas C111-glutathionylation inhibits the association of monomers (decreased kon).

SOD1 represents a common factor in both familial and sporadic cases of ALS (Pasinelli and Brown, 2006; Renton et al., 2014). While most familial mutations destabilize SOD1, our work demonstrates the contrasting influence of a phosphomimetic mutation that stabilizes the native dimer and ameliorates toxicity induced by an aggressive disease mutation. Therefore, stimulating SOD1 phosphorylation in motor neurons or developing drugs to mimic the effects of T2D mutation may offer promising new directions for development of therapeutic strategies. The inhibition of small oligomer formation for disease-linked proteins represents a pharmaceutical approach broadly applicable to many neurodegenerative disorders (Haass and Selkoe, 2007; Kirkitadze et al., 2002). For SOD1, oligomerization and aggregation are proportional to the fraction of monomers (Khare et al., 2004). Researchers have developed various strategies to stabilize SOD1 dimer, including co-expression of chaperones and small molecule binding (Bruening et al., 1999; Limpert et al., 2013; Naik et al., 2012). T2D-SOD1 features a significantly reduce the population of monomers and oligomers, which underlies its beneficial effect to motor neuron-like cells. Hence, identification of T2D as a stabilizing mutation suggests new avenues for therapy development in currently incurable ALS.

EXPERIMENTAL PROCEDURES

Modeling of phosphorylated SOD1 and phosphomimetic mutants

All-atom discrete molecular dynamics (DMD) is a molecular dynamics algorithm that utilizes discrete step functions in place of continuous potentials to describe interaction energies between particles in a simulation (Ding et al., 2008; Proctor et al., 2011). The all-atom protein model features explicit modeling of all heavy atoms, polar hydrogens, and a reaction-based hydrogen bonding potential. We obtained the known structure of SOD1 from the Protein Data Bank (PDB: 1SPD, 1UXM) and performed computational mutagenesis using the Eris suite (Yin et al., 2007a, b). We made no structural adjustment to residues participating in metal-binding or disulfide bond interactions. Following mutation, we minimized the structure using an iterative relaxation and equilibration protocol (Proctor et al., 2011). We performed single-temperature and replica exchange DMD simulations of the optimized T2D-SOD1 structure. In the monomer species, we used 12 replicas for a simulation length of 100 ns at temperatures of 0.5 (∼252 K), 0.52 (∼262 K), 0.54 (∼272 K), 0.56 (∼282 K), 0.58 (∼292 K), 0.60 (∼302 K), 0.62 (∼312 K), 0.64 (∼322 K), 0.66 (∼332 K), 0.68 (∼343 K), 0.70 (∼353 K), and 0.72 (∼363 K) kcal mol−1. In the dimer species, we used 16 replicas for a simulation of 50 ns at temperatures of 0.48 (∼242 K), 0.495 (∼249 K), 0.51 (∼257 K), 0.525 (∼264 K), 0.54 (∼272 K), 0.555 (∼280 K), 0.57 (∼287 K), 0.585 (∼295 K), 0.60 (∼302 K), 0.615 (∼310 K), 0.63 (∼317 K), 0.645 (∼325 K), 0.65 (∼327 K), 0.67 (∼337 K), 0.69 (∼347 K), and 0.71 (∼357 K) kcal (mol kB)−1. While the temperatures used in MD simulations do not equate directly with physical temperatures, they can be used to evaluate relative changes in physical temperature. We combined replica trajectories using weighted histogram analysis method (WHAM) (Kumar et al., 1992). We utilized the MMTSB tool to calculate the constant volume heat capacity (Feig et al., 2004) and analyzed energetic states and molecular interactions as described previously (Proctor et al., 2011).

Cloning, expression, and purification of SOD1

We produced the SOD1 mutant vectors (T2D-SOD1, A4V–SOD1 and T2D/A4V–SOD1) with a Stratagene mutagenesis kit (Agilent Technologies, CA) following the provided protocol. Expression, purification, and separation of modified populations were carried out as described previously (Goscin and Fridovich, 1972; Wilcox et al., 2009).

Circular dichroism (CD) and thermal melting points analysis

We collected CD spectroscopy data using a Chirascan Plus instrument (Applied Photophysics, MA). We dialyzed protein samples against 10 mM phosphate buffer (pH 7.4) and diluted them to 0.2 mg/mL for analysis. CD spectra were measured from 260 nm to 185 nm at 20°C with 50,000 readings taken at each 0.5 nm increment. Melting curves were monitored from 20°C to 94°C at 205 nm. The apparent melting temperature was determined by fitting to a two-state model (Zhu et al., 2013).

Size exclusion chromatography

We prepared the samples (8.8 µM SOD1 in 20 mM Tris and 150 mM NaCl, pH 7.4) and incubated them at 37°C for 0 hr, 24 hr, or 7 days. After incubation the samples were analyzed using a Superdex 200 10/300 GL column (GE Healthcare, PA) described previously (Redler et al., 2011).

Surface plasmon resonance (SPR)

SOD1 samples were biotinylated on one subunit using the EZ-link NHS-LC-LC-biotin linker (Life Technologies, CA) (Khare et al., 2004). We then dialyzed the biotinylated SOD1 into PBS buffer (40 mM phosphate, 150 mM NaCl, pH 7.4) before loading onto a SA chip (Biorad, CA). Dimer dissociation was monitored for WT-SOD1, T2D-SOD1 and T2D/A4V–SOD1 samples using the Proteon XPR36 (Bio-Rad, CA) at 25 °C. The dissociation reaction was initiated by flowing PBS buffer with 0.005% Tween 20 over the SA chip. Each SPR curve is an average of six independent measurements Data was analyzed as describe previously (Redler et al., 2011).

Crystallization, data collection, and model building

Crystallization of T2D was accomplished using a hanging drop vapor diffusion method against a well buffer. T2D was crystallized in a buffer of 2 M (NH4)2SO4, 0.1 M Sodium Cacodylate, 20% MPD and 10 mM ZnCl2 (pH 6.5) after a period of approximately 5 months.

The T2D crystals were transferred into the well buffer plus 20% glycerol as cryoprotectant and then flash-frozen in liquid nitrogen. The X-ray diffraction data of T2D crystal were collected at 100K on Beamline X29 at Brookhaven National Laboratory. We processed the raw data with HKL2000 (Otwinowski and Minor, 1997). The structure of SOD T2D was solved by molecular replacement method (Navaza and Saludjian, 1997). We then performed the refinement with Program Refmac5 in CCP4 Suite (Murshudov et al., 2011). The resulted model was rebuilt by program O (Jones et al., 1991) and further refined by Chiron (Ramachandran et al., 2011).

Cell culture, transfection, and cell viability assay

We maintained the NSC-34 cells in Dulbecco’s Modified Eagle Medium growth medium containing 10% FBS (Cashman et al., 1992). We plated approximately 5000 NSC 34 cells in one well of 24-well format plate with differentiation medium (1% FBS+NEAA+10 µM Retinoid Acid). After two days of differentiation, we transfected cells with 2 µg of pCI neo WT SOD1 or pCI neo SOD1 mutants using Lipofectamine 3000 following the manufacturer’s instruction. We performed propidium iodide (for dead cells) and Hoechst (for all cells) staining (Fisher Scientific, PA) 3 days post transfection without fixation (Latt and Stetten, 1976). We fixed the cells 24 hours post-transfection for immunostaining. For Western blot, we used approximately 20,000 cells per well of 6-well format and collected the cells 2 days post-transfection.

Immunoblotting and immunostaining

We performed Western blotting as previously described (Tao et al., 2011). Briefly, cell lysates were denatured in SDS loading buffer, resolved by 18% or 4–20% SDS-PAGE and transferred to a PVDF membrane. Then we incubated the membranes with antibodies against cCaspase3 (Cell Signaling Technology, MA), SOD1 (Calbiochem, MA) and GAPDH, followed by secondary antibody incubation (Moore et al., 1998). The protein bands were visualized with an Odyssey infrared imaging system (LI-COR Biosciences, NE). The expression level of cCaspase 3 was normalized using ImageJ.

Supplementary Material

1
2

Highlights.

  • Stabilizing SOD1 increases the viability of motor neuron-like cells

  • Crystal structure of the stabilizing mutant T2D-SOD1 is reported

  • Biophysical characterizations of T2D-SOD1 reveal stabilizing role of T2-phosphorylation

In Brief.

SOD1 destabilization and misfolding are associated with the fatal neurodegenerative disease ALS. Fay and Zhu et al. report the discovery of a phosphomimetic mutation, T2D, which stabilizes SOD1 and rescues the cytotoxic effect of an ALS-associated mutant.

Acknowledgments

We thank Dr. Michael Caplow, Dr. Feng Ding and Dr. Marino Convertino for valuable discussions. We also thank Dr. Rachel Redler and Dr. Lanette Fee for outstanding support of the study. This work was supported by NIH Grant R01GM080742 (to N.V.D.). E.A.P. was supported by a Ruth L. Kirschstein National Research Service Award (F31AG039266) from the National Institute on Aging.

Footnotes

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ACCESSION NUMBERS

The accession numbers for the structures reported in this paper are PDB: 5K02

AUTHOR CONTRIBUTIONS

J.M.F., C.Z., and N.V.D. designed research; J.M.F., C.Z., E.A.P., and Y.T. performed research; J.M.F., W.C., and H.K. obtained crystal structure; C.Z., E.A.P. and J.M.F. analyzed the data; C.Z., J.M.F., E.A.P. and N.V.D. wrote the paper.

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Associated Data

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Supplementary Materials

1
NIHMS813878-supplement-1.pdf (1.6MB, pdf)
2
NIHMS813878-supplement-2.pdb (178.3KB, pdb)

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