A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation

Abstract

The misfolding and mutation of Cu/Zn superoxide dismutase (SOD1) is commonly associated with amyotrophic lateral sclerosis (ALS). SOD1 can accumulate within stress granules (SGs), a type of membraneless organelle, which is believed to form via liquid-liquid phase separation (LLPS). Using wild-type, metal-deficient, and different ALS disease mutants of SOD1 and computer simulations, we report here that the absence of Zn leads to structural disorder within two loop regions of SOD1, triggering SOD1 LLPS and amyloid formation. The addition of exogenous Zn to either metal-free SOD1 or to the severe ALS mutation I113T leads to the stabilization of the loops and impairs SOD1 LLPS and aggregation. Moreover, partial Zn-mediated inhibition of LLPS was observed for another severe ALS mutant, G85R, which shows perturbed Zn-binding. By contrast, the ALS mutant G37R, which shows reduced Cu-binding, does not undergo LLPS. In addition, SOD1 condensates induced by Zn-depletion exhibit greater cellular toxicity than aggregates formed by prolonged incubation under aggregating conditions. Overall, our work establishes a role for Zn-dependent modulation of SOD1 conformation and LLPS properties that may contribute to amyloid formation.

Keywords: SOD1; aggregation; amyotrophic lateral sclerosis (ALS); liquid-liquid phase separation (LLPS); molecular dynamics (MD) simulation.

Figure 1. The structure of SOD1

1. Human SOD1 amino acid sequence comprised 8‐beta sheets (highlighted in blue) connected by seven loop regions (in red); Zn‐binding loop IV and electrostatic loop VII highlighted in purple.

2. Cartoon representation of SOD1 monomer highlighting loops IV and VII (Coulombic surface coloring: red = negative, white = neutral, blue = positive).

3. Schematic representation of SOD1 variants used in the study. WTSOD1^2SH is the disulfide‐reduced holo form with both metal ion cofactors Zn and Cu. ApoSOD1^2SH is the disulfide‐reduced de‐metalated form of WTSOD1^2SH lacking both metal cofactors. ZnSOD1^2SH and CuSOD1^2SH are the only Zn‐ and Cu‐bound forms of the disulfide‐reduced monomer.

Figure 2. ApoSOD1^2SH undergoes liquid–liquid phase separation

1. SOD1^2SH undergoes LLPS on the removal of metal ion cofactors.

2. DIC microscopic image of WTSOD1^2SH diluted to 100 μM in HEPES buffer, pH 7.4 and 100 mM NaCl, and incubated 37°C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 μm; inset shows Alexa Fluor 488 maleimide‐labeled WTSOD1^2SH; scale bar: 5 μm.

3. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD1^2SH (at concentrations ranging from 25 to 200 μM does not undergo LLPS in the presence or absence of heparin, LLPS inducer; data are represented as mean ± SD (from three biological replicates).

4. DIC microscopic image of ApoSOD1^2SH under condensate inducing conditions (37°C, 180 RPM) at a concentration of 100 μM incubated 100 mM NaCl; scale bar:100 μm (inset on bottom left shows Alexa Fluor 488 maleimide‐labeled protein condensates; scale bar: 5 μm.

5. Fluorescent microscopic images of Alexa Fluor 488 maleimide‐labeled ApoSOD1^2SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37°C) in the absence (left) and presence (right) of heparin at 30 min time point; scale bar: 100 μm.

6. Solution turbidity measurements with ApoSOD1^2SH (absorbance at 600 nm) show that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS; data are represented as mean ± SD (from three biological replicates).

7. Comparison of ApoSOD1^2SH droplet numbers per microscopic field of view (view area 0.001963 mm²) calculated from five different images of droplets incubated with and without heparin at 2 h time point. Data are represented as mean ± SD. Statistical significance was established using an unpaired nonparametric t‐test (****P < 0.0001).

8. Liquid nature of ApoSOD1^2SH showing droplet fusion and surface wetting; scale bar: 5 μm.

9. Amplitude normalized FCS curves of ApoSOD1^2SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τ_D (as shown by the arrow) translates to a decrease in (D; please refer to equation (2) in Materials and Methods section).

10. Scheme depicting that presence of Zn and not Cu in a preincubation mixture inhibits LLPS.

11. DIC microscopic images of ApoSOD1^2SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets; scale bar: 100 μm.

12. Solution turbidity plot (absorbance at 600 nm) of ApoSOD1^2SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose‐dependent manner for Zn while no significant change is observed in presence of Cu; data are represented as mean ± SD (from three biological replicates).

Source data are available online for this figure.

Figure 3. ALS mutants with compromised Zn binding undergo LLPS

1. DIC microscopic images of I113T SOD1^2SH (top) and G85R SOD1^2SH (bottom) droplets incubated in the absence and presence of Zn and Cu, respectively (scale bar: 100 μm); insets on bottom left show Alexa Fluor 488 maleimide‐labeled protein condensates for I113T SOD1^2SH and G85R SOD1^2SH, respectively; scale bar: 5 μm. Droplet dissolution was observed for I113T SOD1^2SH on the addition of Zn while droplets persisted on the addition of Cu. Red arrowhead indicates fusion of liquid droplets.

2. Amplitude normalized FCS curves of I113T SOD1^2SH (in green) and G85R SOD1^2SH (in gray) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP, and light variant indicates DP.

3. On metal cofactor removal, ApoG37R SOD1^2SH formed condensates, which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right); scale bar: 100 μm.

Source data are available online for this figure.

Figure 4. Zn drives disorder to order transition in SOD1

1. Scheme shows Zn‐dependent disorder to order transition in monomeric SOD1.

2. Plot of the log_e of the hydrodynamic radius (r _H) versus the log_e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y‐axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (gray dashed) data has a slope of 0.57 ± 0.02 and a y‐axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the r _H of all SOD1^2SH variants with (solid shapes) and without Zn (hollow shapes). The hollow gray circle of G85R SOD1^2SH is directly under hollow green I113T SOD1^2SH in the log–log plot.

3. Variation of diffusion coefficients of de‐metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD1^2SH and ApoG37R SOD1^2SH has a higher binding affinity to Zn than ApoI113T SOD1^2SH and ApoG85R SOD1^2SH). Data are represented as mean ± SD (from three biological replicates).

4. Disordered/ extended conformation content decreases in ApoSOD1^2SH and ApoI113T SOD1^2SH with the addition of Zn calculated from FTIR spectra; data are represented as mean ± SD (from three biological replicates).

Source data are available online for this figure.

Figure 5. Characterization of SOD1^2SH variants and molecular interactions in the condensed phase using molecular dynamics simulations

1. A, B

   (A) Per‐residue RMSF profiles of ApoSOD1^2SH & its mutants computed from 5 μs simulations performed for each variant. (B) Per‐residue RMSF profiles of ApoSOD1^2SH, ApoSOD1^S‐S, ZnSOD1^S‐S, and ZnSOD1^2SH computed from 5 μs simulations performed for each variant. Error bars in A and B correspond to standard errors, which are estimated by blocks averaging over 10 (500 ns each) blocks.

2. C

   Salt‐bridge analysis of ApoG85R SOD1^2SH (dist. cutoff < 0.5 nm = salt bridge).

3. D

   Snapshots from the ApoG85R SOD1^2SH trajectory showing the formation of non‐native salt bridges, which are likely to be detrimental for Zn‐binding.

4. E

   Density profile of ApoSOD1^2SH and its loop‐deletion variants computed from the CG slab simulations. The condensed phase is centered in the middle of the simulation box.

5. F

   Slab snapshots from CG simulations of ApoSOD1^2SH and its variants at 275 K (blue beads represent loop IV, red beads represent loop VII, and orange beads represent the other domains).

6. G

   Pairwise intermolecular contact formation (normalized by maximum probability) in the condensed phase as a function of residue index.

7. H

   Saturation concentration measured in the CG phase coexistence simulations for ApoSOD1^2SH and its variants including ALS mutants at 275 K. The error bars correspond to SD, which are estimated from block averages over four blocks.

Source data are available online for this figure.

Figure 6. Maturation of liquid droplets precedes aggregation

1. Cartoon scheme showing the maturation of protein droplets with time (region1: diffused region outside droplet, region 2: low‐intensity portion inside the droplet, region 3: highly intense portion inside droplet appears during maturation) at different time points.

2. Diffusion coefficient of regions 1 and 2 (within droplet) was measured using point FCS as shown in the scheme.

3. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488‐labeled ApoSOD1^2SH droplets with time, samples were incubated at 37°C up to ~ 25 h. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 h, and > 24 h; images below show magnified scale bar: 5 μm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows the coexistence of liquid and solid phases inside droplet; scale bar: 10 μm. Intensity plot (bottom right) shows the distribution of high‐intensity region within matured droplet.

4. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x‐axis label) indicates the time after incubation required for droplet formation. Data are shown mean ± SD (from three biological replicates). Statistical significance was measured using paired t‐test; ns denotes nonsignificant; **P < 0.01; ***P < 0.001.

5. ThT fluorescence assay plot showing aggregation kinetics of all SOD1^2SH variants. ApoSOD1^2SH, I113T SOD1^2SH, and G85R SOD1^2SH show high ThT fluorescence.

6. AFM micrographs of ApoSOD1^2SH, I113T SOD1^2SH, and G85R SOD1^2SH show fibrillar aggregates after prolonged incubation at 37°C, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD1^2SH, I113T SOD1^2SH, and G85R SOD1^2SH incubated with Zn (at 1:5 protein:Zn molar ratio) show the presence of oligomers for ApoSOD1^2SH, I113T SOD1^2SH and short fibrils for G85R SOD1^2SH (right panel); scale bar: 200 nm.

7. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 μM ApoSOD1^2SH, I113T SOD1^2SH, and G85R SOD1^2SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as a positive control. Data are shown as mean ± SEM (from three biological replicates). P values were determined by two‐tailed unpaired t tests; *P < 0.05, **P < 0.01; ***P < 0.001, ****P < 0.0001.

8. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 μM ApoSOD1^2SH condensates, I113T SOD1^2SH condensates, G85R SOD1^2SH condensates, ApoSOD1^2SH fibrils, I113T SOD1^2SH fibrils, and G85R SOD1^2SH fibrils, respectively (see Materials and Methods for details). High cytotoxicity was observed for SOD1^2SH condensates.

Source data are available online for this figure.

Figure 7. A schematic describing the mechanism by which Zinc regulates LLPS and aggregation of SOD1

In the absence of Zn, SOD1 exhibits conformational disorder (instability) in the loop regions (IV/VII), which promotes LLPS and the subsequent maturation of condensates into aggregates. Upon binding to Zn, the loop regions adopt a rigid conformation, which inhibits LLPS.