DnaJB1 chaperone inhibits tau aggregation by recognizing its N-terminus

A network of protein folding and degradation machineries maintains protein homeostasis by preventing the accumulation of misfolded proteins and by facilitating their clearance. These systems are also crucial for the inhibition of protein aggregation in neurodegenerative diseases where misfolded proteins often aggregate into β-rich amyloid fibrils. How these machineries selectively recognize pathological aggregates over normal conformations remains unclear. Here, we present the molecular logic for how a Hsp70 co-chaperone from the J-domain protein family, DnaJB1, binds pathological aggregates of the microtubule-associated protein tau through the recognition of the flexible N-terminus that comprises the disordered fuzzy coat of fibrils. We show that this interaction contributes to the regulation of tau assembly in cellular models of tau aggregation and depends on the presence of the negatively charged residues. We determined that DnaJB1 inhibits tau aggregation in vitro through these interactions, and found that this weak, transient binding can be enhanced by the presence of polyanionic factors such as heparin. As prospective client-binding sites, we identified the charged hinge between the two β-sandwich C-terminal domains I and II, as well as the conserved J-domain of this chaperone. This work presents novel biochemical and structural insights into how the molecular chaperone DnaJB1 recognizes full-length forms of tau protein in a pathological context. Description of deposited files Source Data: All analyzed cell-based aggregation, ThT, ITC, NMR, MST, SEC, and XL-MS data, as well as uncropped western blot membranes and SDS-PAGE gels. Plasmid maps: Annotated plasmid maps of all constructs used in this study. Cell biosensors: Raw flow cytometry and fluorescent microscopy files (Figures 1, 2, 4, and Supplementary Figures 2, 6). ThT: Raw (not baseline corrected) Thioflavin T (ThT) aggregation assay files (Figure 2 and Supplementary Figure 2). MST: Raw microscale thermophoresis (MST) binding assay files (Supplementary Figure 3). ITC: Raw Isothermal titration calorimetry (ITC) binding assay files (Figure 3 and Supplementary Figures 3, 4). NMR: 1H–15N heteronuclear single quantum coherence spectroscopy (HSQC) NMR spectra acquired to investigate interactions of 15N-labeled tau1-243 with unlabeled DnaJB1 in solution. The directory name indicates the protein concentrations used during titration. Each directory includes files that contain the raw NMR data (fid), the acquisition parameters (procpar), a log file (log), macros to convert from Agilent format to NMRPipe format (fid.com) and to process the data with NMRPipe (jrhstgood.com), an intermediate processing file (hsdirt.ft), and the processed NMR spectrum in NMRPipe format (hsdirt.ft2) (Figure 3 and Supplementary Figure 5). XL-MS: xQuest/xProphet prefiltered search results from cross-linking mass spectrometry (XL-MS) experiments, and .pdb model of full-length DnaJB1 dimer made with AlphaFold2 multimer v3 based on PDB ID 3AGY that was used to map inter-protein XL-MS hits. Residue positions for DnaJB1 in xQuest/xProphet search results files are shifted +2 relative to DnaJB1's sequence available under UniProt ID P25685-1. Residue positions in the associated manuscript and in the Source Data file have been corrected appropriately to match the UniProt ID P25685-1 sequence. Residue positions for tau protein refer to 2N4R isoform, available under UniProt ID P10636-8. DMTMM datasets uploaded here are prefiltered (original xQuest/xProphet output) and include hits incompatible with the used cross-linker chemistry: K-K, D-D, E-E. Compatible cross-links are only K-D and K-E (Figure 5 and Supplementary Figure 7). Gels and blots: Unmodified and uncropped western blot membranes and SDS-PAGE gels (Supplementary Figures 1, 5, 7).