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).蛋白质折叠与降解机器构成的网络通过阻止错误折叠蛋白质的积累并促进其清除，维持细胞内的蛋白质稳态（protein homeostasis）。该网络在神经退行性疾病中同样对抑制蛋白质聚集发挥关键作用——此类疾病中错误折叠的蛋白质通常会聚集形成富含β折叠的淀粉样原纤维（β-rich amyloid fibrils）。目前，此类机器如何选择性识别病理状态的聚集体而非正常构象的蛋白质，仍是尚未阐明的科学问题。本研究阐明了J结构域蛋白家族（J-domain protein family）的热休克蛋白70（Hsp70）辅助伴侣蛋白DnaJB1，通过识别构成淀粉样原纤维无序模糊外套层的柔性N端区域，结合微管相关蛋白tau（microtubule-associated protein tau，简称tau）病理聚集体的分子逻辑。我们证实，该相互作用在tau聚集的细胞模型中参与调控tau的组装过程，且其依赖于带负电荷残基的存在。我们进一步确定，DnaJB1可通过上述相互作用在体外抑制tau聚集，并发现这种弱瞬时结合可被肝素（heparin）等聚阴离子因子增强。我们还鉴定出该分子伴侣的两个β折叠三明治C端结构域I与II之间的带电铰链区，以及其保守的J结构域，作为潜在的底物结合位点。本研究为分子伴侣DnaJB1在病理状态下如何识别全长tau蛋白，提供了全新的生化与结构学见解。 已存档文件说明 源数据：本研究中所有经分析的细胞聚集实验、硫黄素T（ThT）、等温滴定量热法（ITC）、核磁共振波谱法（NMR）、微量热泳动（MST）、尺寸排阻色谱（SEC）及交联质谱（XL-MS）数据，以及未裁切的蛋白质免疫印迹膜和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶。 质粒图谱：本研究中使用的全部构建体的注释型质粒图谱。 细胞生物传感器数据：原始流式细胞术与荧光显微镜文件（对应图1、2、4及补充图2、6）。 硫黄素T检测数据：未经过基线校正的原始硫黄素T（ThT）聚集检测文件（对应图2及补充图2）。 微量热泳动检测数据：原始微量热泳动（MST）结合检测文件（对应补充图3）。 等温滴定量热法检测数据：原始等温滴定量热法（ITC）结合检测文件（对应图3及补充图3、4）。 核磁共振波谱检测数据：用于研究15N标记的tau1-243与未标记DnaJB1在溶液中相互作用的1H–15N异核单量子相干谱（HSQC）核磁共振波谱。目录名称标注了滴定过程中使用的蛋白质浓度。每个目录包含以下文件：原始核磁共振数据（fid）、采集参数文件（procpar）、日志文件（log）、将安捷伦格式转换为NMRPipe格式的宏命令（fid.com）、使用NMRPipe处理数据的宏命令（jrhstgood.com）、中间处理文件（hsdirt.ft），以及NMRPipe格式的已处理核磁共振谱图（hsdirt.ft2）（对应图3及补充图5）。 交联质谱检测数据：交联质谱（XL-MS）实验的xQuest/xProphet预过滤搜索结果，以及基于PDB ID 3AGY、使用AlphaFold2 multimer v3构建的全长DnaJB1二聚体的.pdb模型，该模型用于映射蛋白间交联质谱匹配位点。xQuest/xProphet搜索结果文件中DnaJB1的残基位置相较于UniProt ID P25685-1对应的DnaJB1序列偏移了+2。相关论文及源数据文件中的残基位置已进行适当校正，以匹配UniProt ID P25685-1的序列。tau蛋白的残基位置对应2N4R亚型，该亚型的UniProt ID为P10636-8。本处上传的DMTMM数据集为预过滤后的文件（原始xQuest/xProphet输出结果），包含与所用交联剂化学反应不兼容的匹配位点：K-K、D-D、E-E。兼容的交联仅为K-D与K-E（对应图5及补充图7）。 凝胶与印迹数据：未修改、未裁切的蛋白质免疫印迹膜和SDS-PAGE凝胶（对应补充图1、5、7）。