ClpL is an autonomous disaggregase. (A) Domain organizations of ClpL, ClpG, ClpB. All AAA+ proteins consist of two AAA domains (AAA1, AAA2), a coiled-coil middle-domain (M) and diverse N-terminal domains (N). ClpG additionally harbors a disordered C-terminal extension (CTE). (B) Left: Luciferase disaggregation activity (% refolding of aggregated Luciferase/min) of Lm ClpL was determined. Right: Relative Luciferase disaggregation activities of indicated disaggregation systems were determined. KJE: DnaK/DnaJ/GrpE. The disaggregation activity of Lm ClpL was set to 1. (C) GFP disaggregation activities (% refolding of aggregated GFP/min) of indicated disaggregation systems were determined. (D) Occurrence of ClpB and ClpL disaggregases in selected Gram-positive bacteria. Loss of heat resistance upon clpL deletion in bacteria harboring solely ClpL is indicated. n.d.: not determined. (E) Relative Luciferase and MDH disaggregation activities (each: % regain of enzymatic activity/min) of L. gasseri (Lg) ClpL. Disaggregation activities of Lm ClpL were set to 1. Shown is a boxplot (B) or data points and mean ± SD (B/C/E), n ≥ 3. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.

Specific molecular features separate ClpL from ClpB/DnaK. (A) Incubation of Luciferase-YFP at 46°C only leads to unfolding of the Luciferase moiety and the formation of mixed aggregates including folded YFP. Unfolding of YFP during disaggregation of aggregated Luciferase-YFP reports on threading power. (B) Aggregated Luciferase-YFP was incubated in presence of indicated disaggregation machineries and YFP fluorescence was recorded. Initial YFP fluorescence was set at 100%. (C/D) Melting temperatures of ClpL, Lm ClpB and Lm DnaK were determined in presence of indicated nucleotides by SYPRO®Orange binding (C) or nanoDSF (D). Shown are mean curves ± SD (B) or data points and mean ± SD (C/D), n ≥ 3. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.

The ClpL NTD is essential for disaggregation activity. (A) Disaggregation activities of ClpL and ΔN-ClpL towards aggregated Luciferase and MDH (each: % regain enzymatic activity/min) were determined. The activity of ClpL was set to 1. (B) ATPase activities of ClpL and ClpL-ΔN were determined. The ATPase activity of ClpL was set to 1. (C) E. coli ΔclpB cells harboring plasmids for constitutive expression of Luciferase and IPTG-controlled expression of indicated disaggregases were grown at 30°C to mid-logarithmic growth phase and heat shocked to 46°C for 15 min. Luciferase activities prior to heat shock were set to 100%. The regain of Luciferase activity was determined after a 120 min recovery period at 30°C. 10/100/500: μM IPTG added to induce disaggregase expression. p: empty vector control. (D) E. coli ΔclpB cells harboring plasmids for expression of indicated disaggregases were grown at 30°C to mid-logarithmic growth phase and shifted to 49°C. Serial dilutions of cells were prepared at the indicated time points, spotted on LB plates and incubated at 30°C. 10/100/500: μM IPTG added to induce disaggregase expression. p: empty vector control. (E) Quantification of data from (D). (F) Domain organization of the ClpL-ClpB chimera LN-ClpB*. ClpB* harbors the K476C mutation in its M-domain, abrogating ATPase repression. (G) Relative Luciferase disaggregation activities (% refolded Luciferase/min) of indicated disaggregation machineries were determined. The disaggregation activity of ClpL was set to 1. Shown are mean curves and data points (E) or data points and mean ± SD (A/B/C/G), n ≥ 3. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.

Molecular basis of ClpL NTD binding to protein aggregates. (A) AlphaFold2 model of ClpL NTD. The colour code depicts the calculated confidence of the prediction (pLDDT). Residues that potentially participate in the formation of small hydrophobic cores (α1/2 (cyan) and α2/β1,2 (magenta)) are indicated. (B) Secondary structure of ClpL-NTD as determined by NMR using secondary chemical shifts (Cα, Cβ). Secondary structure elements determined from NMR and from the AlphaFold prediction are indicated below the histogram. The predicted α1-helix only transiently forms in isolated solution context, which is confirmed by further NMR analysis (Fig. S5E). (C) Composition of ClpL NTDs. The frequencies (%) of individual amino acids represent the ratio of the number of a particular residue and the total length of respective NTDs (L. monocytogenes, Staphylococcus aureus, S. pneumoniae, Lactobacillus plantarum, Oenococcus oeni, Lactobacillus rhamnosus, Streptococcus suis). The average frequency of each amino acid in the total bacterial proteomes is given as reference (Bogatyreva et al, 2006). (D) Localization of patches A-E, consisting of aromatic and N/Q residues are indicated. (E/F) Luciferase and MDH disaggregation activities (% refolded enzyme/min) of ClpL WT and indicated patch mutants were determined. The disaggregtion activity of ClpL was set to 1. (G) ClpG, ClpL and indicated ClpL mutants were incubated with aggregated MDH in presence of ATPγS. The extend of aggregate binding was determined by co-sedimentation upon centrifugation and quantifications of chaperone levels in soluble and insoluble fractions. Shown are data points and mean ± SD (C/E/F/G), n ≥ 3. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.

Multiple ClpL NTDs are required for disaggregation activity. (A) Varying the ratio of LN-ClpB* and ΔN-ClpB* leads to formation of mixed hexamers with diverse numbers of NTDs. (B) Luciferase disaggregation activities (% refolded Luciferase/min) of mixed LN-ClpB*/ΔN-ClpB* hexamers were determined and compared with curves calculated from a model (black to grey), which assumes that a mixed hexamer only displays disaggregation activity if it contains the number of NTDs indicated. Mixing ratios are indicated as number of ΔN-ClpB* in a hexamer. (C/D) Luciferase disaggregation activities of LN-ClpB* (C) and ClpL (D) were determined in absence and presence of an excess of isolated NTD as indicated. Disaggregation activities determined in NTD absence were set as 100%. Shown are data points (B) or data points and mean ± SD (C/D), n ≥ 3. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.

Stabilizing ClpL ring dimers strongly reduces disaggregation activity. (A) AlphaFold2 model of Lm ClpL ring dimers. Positions of individual domains are indicated. (B) Negative stain EM of Lm ClpL. 2D class averages revealing single ring hexamers or heptamers, ring dimers and tetramers of rings are indicated. The scale bar is 100 nm. (C) Populations (%) of diverse ClpL assembly states based on 2D class averages were determined for Lm ClpL WT and indicated mutants and Lg ClpL. Evaluated particles: nWT = 5233, nΔN = 18314, nΔM = 1900, nE352A = 14215, nF354A = 7074, nLg WT = 11765). (D/E) Disaggregation activities of ClpL WT and indicated M-domain (M) mutants towards aggregated Luciferase (D) and MDH (E) (each: % regain enzymatic activity/min) were determined. The activity of ClpL was set to 1. (F) Model of ClpL ring dimers. Positions of T355 residues in interacting M-domains are depicted. Crosslinking of ClpL T355C was achieved by DTT removal and further incubation at RT as indicated. The formation of crosslinked ClpL T355C dimers was monitored by SDS-PAGE and Coomassie-staining. Addition of 10 mM DTT reversed the disulfide bonds. (G) Luciferase disaggregation activities of reduced ClpL WT (assay control) and oxidized ClpL WT (treatment control) or ClpL T355C were determined in absence of DTT (-DTT). Oxidized variants (WT and T355C) were additionally preincubated with 10 mM DTT for 30 min and tested for disaggregation activity in presence of DTT (+DTT). The factor of increase in disaggregation activity upon reduction (+DTT) is indicated (right). Shown are data points and mean ± SD (D/E/G) or only mean ± SD (G), n ≥ 3. Standard deviations for the activity gain factors (G) have been propagated from disaggregation activity standard deviations. Statistical Analysis: One-way ANOVA, Welch’s Test for post-hoc multiple comparisons. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. n.s.: not significant.