Structural basis for mutation-induced destabilization of profilin 1 in ALS

Abstract

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.

Keywords: X-ray crystallography; amyotrophic lateral sclerosis; profilin 1; protein misfolding; protein stability.

Fig. S1.

A comparison of PFN1 C71G purified from the soluble lysate of Escherichia coli vs. from inclusion bodies. (A) Equilibrium unfolding and (B) thermal denaturation curves (described in Fig. 1) for PFN1 C71G purified from the soluble lysate and inclusion bodies. The apparent melting temperature of PFN1 C71G purified from inclusion bodies (34.62 ± 0.05 °C) is the same as that purified from soluble lysate (34.60 ± 0.03 °C). (C) PFN1 C71G has similar affinities to poly-l-proline as determined by the binding assay described in Fig. 6 irrespective of whether this variant was purified from the soluble lysate or inclusion bodies.

Fig. 1.

ALS-linked mutations destabilize PFN1. Chemical and thermal denaturation studies reveal that ALS-linked variants C71G, M114T, and G118V, but not E117G, are severely destabilized relative to PFN1 WT. (A) Equilibrium unfolding curves for PFN1 WT and ALS-linked variants generated by measuring the intrinsic tryptophan fluorescence of the indicated protein equilibrated in increasing concentrations of urea. Data were processed to obtain the center of mass (COM) of the emission spectrum and then fit to a two-state model for protein folding. The resulting fits are displayed as solid lines. The corresponding thermodynamic parameters obtained from the fitted data are shown in Table 1. (B) Thermal denaturation profiles of PFN1 proteins measured by SYPRO Orange fluorescence as a function of increasing temperature were used to determine the apparent T_m, which is the temperature corresponding to 0.50 fluorescence signal as denoted by the intersection of the dashed lines for each curve.

Fig. S2.

All PFN1 variants unfold by a two-state process. (A–E) PFN1 variants denatured in urea were refolded by diluting the urea. The final concentration of PFN1 in each sample was 10 μM and tryptophan fluorescence was used to monitor folding. The equilibrium transition regions overlay closely for the unfolding and refolding curves, indicating that the unfolding reaction is reversible. Filled and open circles represent unfolding and refolding, respectively. (F) The two-state unfolding of PFN1 observed by intrinsic fluorescence (data from Fig. 1A; Fluor) was verified by CD measurements for PFN1 WT and M114T. The concentration of protein used was 2 μM and 10 μM for tryptophan fluorescence and CD measurements, respectively. The y axis on the left is the mean residue ellipticity at 220 nm (MRE₂₂₀) obtained from CD experiments, whereas the y axis on the right reflects the change in the COM (as shown in Fig. 1). The thermodynamic parameters obtained by fitting the CD data agree well with those obtained from the fluorescence data (Table 1) and are as follows: for WT ΔG° = 7.16 ± 0.11 kcal⋅mol⁻¹, m = 2.36 ± 0.04 kcal⋅mol⁻¹⋅M⁻¹, C_m = 3.03 ± 0.07 M; for M114T ΔG° = 4.35 ± 0.10 kcal⋅mol⁻¹, m = 2.95 ± 0.06 kcal⋅mol⁻¹⋅M⁻¹, C_m = 1.47 ± 0.05 M.

Fig. 2.

ALS-linked PFN1 variants exhibit faster turnover in a neuronal cell line. SKNAS cells transiently transfected with V5-PFN1 constructs were treated with cycloheximide (CHX) for up to 12.5 h, during which time lysates were collected and probed by Western analysis with a V5-specific antibody to assess the rate of PFN1 turnover in cells. (A and B) A representative Western blot analysis of soluble and insoluble fractions from cell lysates demonstrates a decrease in V5-PFN1 protein with time. GAPDH serves a loading control for the soluble fraction. (C) Densitometry analysis of A reveals that the turnover of PFN1 C71G and M114T is significantly faster than that of PFN1 WT. Statistical significance was determined using a two-way ANOVA followed by a Tukey’s post hoc analysis (*P < 0.05, **P < 0.01, ^#P < 0.0001). Error bars represent SEM. WT and E117G, n = 3; G118V, M114T and C71G, n = 4 independent experiments.

Fig. S3.

The turnover of insoluble PFN1 in SKNAS cells. The experiment was carried out as described in Fig. 2, and a representative Western blot analysis of the insoluble fraction is shown in Fig. 2B. The data above reflect the densitometry results from an average of n = 2 (M114T) or n = 3 (C71G and G118V) independent experiments and error bars represent SEM. Each sample was normalized to the PFN1 C71G band corresponding to “time 0.” The turnover of C71G within the insoluble fraction was slower relative to C71G within the soluble fraction (compare this graph to that in Fig. 2C). There was relatively less M114T and G118V in the insoluble fraction compared with C71G, and the small fraction of insoluble G118V persisted throughout the experimental time course.

Fig. S4.

ALS-linked PFN1 variants retain the same secondary structure as PFN1 WT. (A–D) Far UV CD spectra for the indicated PFN1 variant (10 μM) overlaid with CD spectrum for PFN1 WT (10 μM).

Fig. S5.

Analysis of PFN1 proteins by native page and analytical size-exclusion chromatography. (A) PFN1 proteins (10 μg) were subjected to native (Top) or denaturing (Bottom) gel electrophoresis and detected with Coomassie Brilliant Blue stain. The mobility of native PFN1 WT is indicated. PFN1 E117G migrates with a slightly faster mobility than PFN1 WT owing to the addition of a negatively charged amino acid. Misfolded ALS-linked PFN1 variants migrate with slower mobility and form aggregated species that are retained in the stacking gel. This gel is representative of n = 2 experiments using proteins from different purification preparations. (B–F) The indicated PFN1 protein (40 μg) was subjected to analytical size-exclusion chromatography using a Superdex 75 column. A single peak corresponding to the expected elution volume (∼15 mL) for monomeric PFN1 was detected for all PFN1 proteins. The experiments were carried out in duplicate for each variant, indicated by solid (n = 1 experiment) and dashed (n = 2 experiment) lines. The average relative peak area ± the SD is indicated to the right of each curve. Despite equal sample loading, the peak area of PFN1 C71G and M114T is lower than that of WT (within error), consistent with a reduced level of soluble protein for these ALS-linked variants. (G) An overlay of B–F for the n = 1 experiment demonstrates a similar elution profile for all PFN1 proteins.

Fig. 3.

Superimposition of the crystal structures for PFN1 WT, E117G, and M114T. (A and B) The secondary and tertiary structures for PFN1 WT (green), E117G (mustard), M114T chain A (pink), and B (red) are highly superimposable. For each structure, sticks and spheres denote the side chains and van der Waals radii, respectively, for residues at position 114 and 117. Residue 117 is located within a solvent-exposed flexible loop that has no discernible secondary structure, whereas Met114 is located within a β-sheet toward the interior of the protein. (B) A zoomed cartoon representation showing residues within 4 Å of residue 114. The side chains of these residues are indicated as sticks with nitrogen, oxygen, and sulfur atoms indicated in blue, red, and yellow, respectively. The van der Waals radii of the atoms comprising residue 114 are reduced upon mutation of methionine (green and mustard structures) to threonine (red and pink structures).

Fig. S6.

Structural changes induced by the M114T mutation revealed in double difference plots. Double different plots (Left) of WT vs. E117G (A), WT vs. M114T chain A (B), WT vs. M114T chain B (C), and M114T chains A vs. B (D). The Avg-Abs-DD values are plotted as a function of residue number for each structural comparison (Middle); these plots provide an indication for residues that undergo a structural change between the proteins that are being compared. Residues with Avg-Abs-DD values of 0.3 Å or greater are plotted onto the structure (Right) of PFN1 WT (A–C) and PFN1 M114T chain A (D) in green. Residues not used in this analysis are colored black.

Fig. 4.

Structure of actin–PFN1–VASP peptide ternary complex with the actin and poly-l-proline binding residues mapped on PFN1. The X-ray structure of the PFN1 WT (gray)–actin (blue)–poly-l-proline peptide (gold) complex (PDB ID code 2PAV) is shown. Residues reportedly involved in actin binding (V61, K70, S72, V73, I74, R75, E83, R89, K91, P97, T98, N100, V119, H120, G122, N125, K126, Y129, and E130) and poly-l-proline binding (W4, Y7, N10, A13, S28, S30, W32, H134, S138, and Y140) are highlighted in blue and gold, respectively. The sites of ALS-linked mutations investigated in this study are highlighted and labeled in black with side chains displayed as black sticks. Residues involved in actin or poly-l-proline binding that also exhibit Avg-Abs-DD values of 0.3 Å or greater between PFN1 WT and M114T chain B (W4, K126, and S138) are labeled in black (the remaining residues that fulfill this criteria are shown in Fig. S7).

Fig. S7.

Actin and poly-l-proline binding residues exhibit relatively high double difference values. Residues that have Avg-Abs-DD values of 0.3 Å or greater that are also engaged in actin binding (V119, H120, G122, and K126) or poly-Pro binding (W4, Y7, H134, and S138) are mapped onto the structure of PFN1 WT in magenta. All other residues with Avg-Abs-DD values of 0.3 Å or greater are highlighted in green. Residues with Avg-Abs-DD values between chain A and chain B of M114T 0.3 Å or greater (Fig. S6D) were excluded from this analysis. Residues not used in this analysis are colored black.

Fig. S8.

The calculated α-carbon B factors for all PFN1 structures. Cartoon representations of WT (A), E117G (B), and M114T chains A (C) and B (D). Residues are colored according to the α-carbon B factors using the scale shown at the bottom. The average α-carbon B factor for WT, E117G, and M114T chains A and B structures are 30.52, 22.94, 29.47, and 27.33, respectively. Because the average B factor is higher for M114T chain A, M114T chain B was used for structural analyses unless otherwise noted.

Fig. 5.

ALS-linked PFN1 variants retain the ability to bind poly-l-proline. (A) Binding of PFN1 to the poly-l-proline peptide was monitored by measuring the intrinsic tryptophan fluorescence of the indicated PFN1 protein as a function of increasing peptide concentration. The data points were fit using a one-site total binding model in GraphPad Prism and the apparent dissociation constants (K_d) obtained from the fit are shown in Table 1. Note that the concentration of the peptide is reported in terms of [proline] because the peptide stock is supplied as a mixture of poly-l-proline species (Materials and Methods). (B) DSF was performed as described in Fig. 1B in the presence (dashed lines) and absence (solid lines) of 4 mM proline. The presence of proline increases the T_m for all PFN1 proteins used in this study (Table 1), as illustrated here for WT, C71G, and M114T.

Fig. 6.

The binding of PFN1 proteins to G-actin. Polymerization of monomeric rabbit muscle actin (3 μM, 5% pyrene-labeled) was monitored in the presence of increasing concentrations of WT or ALS-linked PFN1 variants and used to derive relative rates of polymerization (n = 3). The variant H120E, which is impaired in binding to actin, fails to suppress spontaneous actin polymerization as effectively as WT PFN1. Although G118V is relatively weak in suppressing actin polymerization, the data did not reach statistical significance. Statistical significance was determined using a two-way ANOVA followed by a Tukey’s post hoc analysis. **P ≤ 0.01 for WT vs. H120E at 7 μM concentration. No other significant comparisons with WT were obtained. Other significant comparisons included C71G vs. H120E and E117G vs. H120E (P ≤ 0.05) at 7 μM concentration. Error bars represent SD.

Fig. 7.

The M114T mutation causes a surface-exposed pocket to expand into the core of the PFN1 protein. (A) Residues are depicted as described in Fig. 3. The van der Waals radii of residues 90, 114, and 18 are in contact in the PFN1 WT structure (Top). These contacts are reduced by the M114T mutation (Bottom) owing to the smaller size of threonine, leading to an enlargement of the surface-exposed pocket. (B) PFN1 WT is shown with a transparent surface and the secondary structure is shown in cartoon representation. The surface pocket volume for PFN1 WT (green) and the cleft volume for PFN1 M114T chain B (red) are depicted as opaque surfaces and were generated using SiteMap. The predicted cavity (blue) for PFN1 C71G (generated using PyMOL) overlays with the M114T void, and unlike the WT and M114T volumes, is not surface-exposed. The insets (Right) show the aforementioned voids for WT (Top), M114T chain B (Middle), and C71G (Bottom).

Fig. S9.

Electrostatic surface potential (ESP) of PFN1 WT and PFN1 M114T. A comparison of the ESP for PFN1 WT (A) and M114T (B) around the surface pocket (for WT) and cleft (for M114T) shown in Fig. 7. Comparison of the ESP was calculated using Maestro (Schrödinger, LLC). The Red_White_Blue color scheme was used to depict the ESP of both surfaces, where red denotes negative, blue denotes positive, and white denotes neutral ESP. The minimum and maximum values are −0.12 and 0.12, respectively. The cleft (boxed region in B) formed by M114T exposes a deeper pocket comprised of hydrophobic residues that would otherwise be buried beneath the surface-exposed pocket (boxed region in A) in PFN1 WT.