Fig 1: Potentiated Hsp104 variants have improved selectivity for disordered substrates in vitro.(A–C) The indicated constructs: RepA1-70-Kaede (A), 6-His-TEV-Kaede (B), or untagged Kaede (C) were incubated with Hsp104 or indicated variants plus ATP or ATP:ATPϒS, and Kaede unfolding was monitored by fluorescence. Values are means ± SEM (n ≥3).(D) Luciferase aggregates were incubated with buffer, Hsp104, or indicated variants (1μM monomer) ±Hsc70 and Hdj2 (0.167μM each). Luciferase activity was normalized to Hsp104WT+Hsc70+Hdj2. Values were compared with corresponding Hsp104WT values using separate one-way ANOVA’s with Dunnett’s multiple comparisons tests (n ≥3, bars represent mean ± SEM: ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Note: the data originally shown in Figures 5D and S4E are shown again here for comparison.(E) ATPase activity of Hsp104 or indicated variants. Values were compared with Hsp104WT by one-way ANOVA with Dunnett’s multiple comparisons test (n ≥3, bars represent mean ± SEM: ns p > 0.05, *p < 0.05, ****p < 0.0001). Note: the data originally shown in Figures S4D and 5C are shown again here for comparison.(F) Assays were performed as in (E) for the indicated variants ±2μM casein. Values were compared to corresponding Hsp104WT alone data or that with casein by one-way ANOVA with Dunnett’s multiple comparisons test as described in (E).(G) Recombinant TDP-43 aggregates were incubated with buffer, Hsp104, or the indicated variants. Insoluble material was quantified by immunoblotting.(H) Quantification of (G). Values are normalized and compared with no Hsp104 by one-way ANOVA with Dunnett’s multiple comparisons test (n ≥3, bars represent means ± SEM: ns p > 0.05, **p < 0.01, ****p < 0.0001). (I and J) Experiments were performed as in (G) and (H), but with α-syn preformed fibrils.
Fig 2: The MD changes orientation as Hsp104 hexamers switch from ATP-bound to ADP-bound states, which alter NBD1:MD interactions(A and B) Left: three out of six protomers (P3 in green, P4 in blue, and P5 in magenta) are shown for the ATP state (A) or ADP state (B). NBD1 is shown in ribbon, and MD is shown in cartoon. The four MD helices (L1, L2, L3, and L4) are indicated by arrows. However, MD helix L3 appears to be a loop in these structures. Right: the NBD1:MD interactions for protomers P3 (green) and P4 (blue) were analyzed using Discovery Studio Visualizer with a 4-Å cutoff distance for the ATP-bound state (AMP-PNP, PDB: 5KNE) (A) or the ADP-bound state (PDB: 5VY8) (B). Hydrophobic, electrostatic (salt bridge), and hydrogen-bond interactions are indicated by red, black, and blue lines, respectively. For the ATP-bound state (A), NBD1:MD interactions were observed between the NBD1 of P3 (green circles) and MD of P4 (blue circles). For the ADP-bound state (B), we show NBD1:MD interactions within P3 (green circles) or within P4 (blue circles) and a unique interaction between R366 and E521 within P5 (purple) for clarity. Intra-subunit NBD1:MD interactions conserved in P3 and P4 are shown in circles with blue and green stripes. Residues involved in intrasubunit NBD1:MD interactions for P3, P4, or P5 are shown in circles with blue and green stripes with an asterisk.(C) NBD1:MD helix L1 interactions with NBD1 in the ATP state. Four major salt-bridge interactions, (1) E190:R419, (2) R194:E412, (3) R353:E427, and (4) R366:D434, are identified on the intersubunit NBD1:MD interface in the presence of AMP-PNP. E190, R194, R353, and R366 are in NBD1 of subunit P3, and R419, E412, E427, and D434 are in MD helix L1 of P4.(D) ATPase activity of Hsp104, Hsp104E190K, and Hsp104R419E (0.25 μM) in the presence of 1 mM ATP at 25°C. Bars represent means ± SEM (n = 4); each replicate is shown as a dot. Ordinary one-way ANOVA Dunnett’s test was performed to compare the ATPase activity of Hsp104 to Hsp104E190K or Hsp104R419E. ns, not significant; ***p = 0.0005.(E) Hsp104 variants (1 μM, monomer) in the presence of ATP/ATPγS (2.5 mM:2.5 mM; black bars) or with Hsc70 (0.167 μM) and DnaJA1 (0.167 μM; pink bars) were incubated with 100 nM (monomeric concentration) chemical-denatured luciferase aggregates for 90 min at 25°C. Buffer serves as the negative control. Bars represent means ± SEM (n = 4); each replicate is shown as a dot. ****p ≤ 0.0001.(F) Survival (%) of Δhsp104 yeast transformed with empty vector (pRS313HSE), Hsp104, Hsp104E190K, or Hsp104R419E after 20 or 30 min of heat shock at 50°C following a pretreatment at 37°C for 30 min. Bars represent means ± SEM (n = 4); each replicate is shown as a dot. ns, not significant; ****p ≤ 0.0001.(G) Δhsp104 yeast from (F) were incubated at 37°C for 30 min to induce Hsp104 expression. Yeasts were then lysed and processed for western blot. 3-Phosphoglycerate kinase 1 (PGK1) serves as a loading control.See also Figure S1.
Fig 3: Rewiring the ATP-specific interprotomer NBD1:MD helix L1 interaction alters Hsp104 collaboration with Hsp70 and Hsp40(A) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM, monomer) in the absence (gray bars) or presence (black bars) of Hsc70 (0.167 μM) and DnaJA1 (0.167 μM). Bars represent means ± SEM (n = 2–4); each replicate is shown as a dot. One-way ANOVA Dunnett’s test was performed to compare Hsp104 to Hsp104 variants at a 95% confidence interval (CI). The luciferase disaggregation and reactivation activity of all tested Hsp104 variants with Hsc70 & DnaJA1 is significantly different from Hsp104 with Hsc70 & DnaJA1 with ****p ≤ 0.0001 (statistics are omitted for clarity).(B) Left panels: spotting assay to evaluate the survival of Δhsp104 yeast transformed with empty vector (no Hsp104) or the indicated Hsp104 variants (WT, wild type) after a 30-min pretreatment at 37°C followed by a 30-min heat shock at 50°C. Δhsp104 yeast that were not heat shocked are shown on the left as a control. Right panels: yeast survival (percentage of WT Hsp104) was quantified. Bars represent means ± SEM (n = 4), and each replicate is shown as a dot. One-way ANOVA Dunnett’s tests were performed to compare Hsp104 variants to no-Hsp104 control (left) or WT (right) at 95% CI. ns, not significant; ***p ≤ 0.001; ****p ≤ 0.0001. One-way ANOVA Tukey tests were performed to make pairwise comparisons between specific Hsp104 variants as indicated (left), ****p ≤ 0.0001.(C) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM, monomer) in the presence of ATP/ATPγS (2.5 mM:2.5 mM) and the absence of Hsp70 and Hsp40. Bars represent means ± SEM (n = 2); each replicate is shown as a dot. One-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to Hsp104 at 95% CI. ns, not significant.(D) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM monomer) in the presence of Ssa1 (0.167 μM) and absence of Hsp40. Bars represent means ± SEM (n = 4); each replicate is shown as a dot. One-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to WT Hsp104 at 95% CI. All variants have significantly reduced activity to work with Ssa1 compared to WT Hsp104 with p ≤ 0.0001 (statistics are omitted for clarity). Alternatively, one-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to no Hsp104. ns, not significant; ****p ≤ 0.0001.(E) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM, monomer) in the presence Ssa1 (0.167 μM) and Sis1 (0.167 μM). Bars represent means ± SEM (n = 4); each replicate is shown as a dot. One-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to WT Hsp104 at 95% CI. ns, not significant. All other variants have reduced activity with p ≤ 0.0001 (statistics are omitted for clarity). Alternatively, one-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to no Hsp104. ns, not significant; ****p ≤ 0.0001.(F) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM, monomer) in the presence of Ssa1 (0.167 μM) and Ydj1 (0.167 μM). Bars represent means ± SEM (n = 4); each replicate is shown as a dot. One-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to WT Hsp104 at 95% CI. All Hsp104 variants have reduced activity with ****p ≤ 0.0001 (statistics are omitted for clarity). Alternatively, one-way ANOVA Dunnett’s test was performed to compare Hsp104 variants to no Hsp104. ns, not significant; ****p ≤ 0.0001.See also Figure S3.
Fig 4: Specific perturbation of ATP-specific NBD1:MD helix L1 contacts yields Hsp104 variants that collaborate selectively with class B Hsp40s(A) Luciferase disaggregation and reactivation activity of the indicated Hsp104 variants (1 μM, monomer) in the presence of: Hsc70 (0.167 μM) and DnaJA1 (0.167 μM; black); Ssa1 (0.167 μM), Ydj1 (0.083 mM), and Sis1 (0.083 μM; pink); Ssa1 (0.167 μM) and Ydj1 (0.167 mM; green); or Ssa1 (0.167 μM) and Sis1 (0.167 μM; purple). Bars represent means ± SEM (n= 2–4); each replicate is shown as a dot.(B) Luciferase disaggregation and reactivation activity of Hsp104 (gray dots), Hsp104R366E (black dots), Hsp104R419E (red dots), or Hsp104D434K (teal dots; 1 μM, monomer) in the presence of various Ssa1 (Hsp70) concentrations in the absence of Hsp40. Curves were fit for absolute EC50 (see STAR Methods and Table S1). Values represent means ± SEM (n = 2).(C and D) Luciferase disaggregation and reactivation activity of Hsp104R366E (1 μM monomer) plus Ssa1 (0.167 μM; black) or Hsp104R419E (1 μM monomer) plus Ssa1 (0.167 μM; red) as a function of Ydj1 (C) or Sis1 (D) concentration. Curves were fit for IC50 of Ydj1 and EC50 of Ydj1 or Sis1 (see STAR Methods and Table S1). Values represent means ± SEM (n = 2).(E and F) Luciferase disaggregation and reactivation activity of Hsp104 (1 μM, monomer) plus Ssa1 (0.167 μM; gray) or Hsp104A503S (1 μM, monomer) plus Ssa1 (0.167 μM; green) as a function of Ydj1 (E) or Sis1 (F) concentration. Curves were fit for IC50 of Ydj1 and EC50 of Ydj1 or Sis1 (see STAR Methods and Table S1). Values represent means ± SEM (n = 2).See also Figures S1 and S2; Table S1.
Fig 5: Hsp104P411S tunes Hsp104 properties(A) W303aΔhsp104 yeast integrated with FUS, α-syn, or TDP-43 were transformed with plasmids encoding Hsp104, indicated variants, or vector. Strains were spotted in serial dilutions on glucose (non-inducing, 6th dilution shown) or galactose (inducing) media. SS were compared with Hsp104WT by one-way ANOVA with Dunnett’s multiple comparisons test (bars represent means ± SEM: ns p > 0.05, ****p < 0.0001).(B) Top-down view of Hsp104 (PDB:5VY8). The inset shows key MD residues in purple sticks.(C) ATPase activity of Hsp104 or indicated variants. Values were compared with Hsp104WT by one-way ANOVA with Dunnett’s multiple comparisons test (n ≥ 3, bars represent mean ± SEM: ns p > 0.05, ***p < 0.001, ****p < 0.0001).(D) Luciferase aggregates were incubated with buffer, Hsp104, or indicated variants (1μM monomer) ±Hsc70 and Hdj2 (0.167μM each). Luciferase activity was normalized to Hsp104WT+Hsc70 and Hdj2. Values were compared with corresponding Hsp104WT values using separate one-way ANOVA’s with Dunnett’s multiple comparisons tests (n ≥3, bars represent means ± SEM: ns p > 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001).(E) Luciferase aggregates were incubated with buffer, Hsp104, or indicated variants (1μM monomer), or indicated mixtures (1μM total monomer). 2×and 3×indicate 2μM or 3μM total monomer, respectively. Values were compared with Hsp104WT by one-way ANOVA with Dunnett’s multiple comparisons test (n ≥3, bars represent means ± SEM: ns p > 0.05, ***p < 0.001).
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