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.