Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells

Abstract The frontotemporal dementia-linked S320F mutation in the microtubule-associated protein tau promotes spontaneous aggregation, yet the structural basis of its amyloidogenesis remains unclear. Using cryo-electron microscopy, we determined the structure of an S320F295-330 tau fibril composed of three parallel chains stabilized by the 306VQIVYK311 amyloid motif, with S320F buried in the fibril core and a unique C322-C322 disulfide linking two protofilaments. Although cysteines are dispensable for fibril formation by isolated peptide fragments in vitro, tau repeat domain constructs containing both C291 and C322 generate more potent seeds in cellular assays. In contrast, the C322S mutation suppresses spontaneous aggregation of S320F tau in cells, and combined C291S and C322S mutations inhibit seeded aggregation in both wild-type and S320F contexts. Systematic alanine mutagenesis coupled with seeding by tauopathy-derived material identifies cysteine residues as critical determinants of tau seeding, comparable in importance to core amyloid motifs. Together, these findings establish cysteines as central chemical regulators of tau aggregation and propagation. Data availability The structural datasets generated during the current study are available in the Electron Microscopy Data Bank repository (https://www.ebi.ac.uk/emdb/) under accession number, EMD-71887 [https://www.ebi.ac.uk/emdb/EMD-71887]. The structural model that was fit into the density is available in the Protein Data Bank under PDB id 9PVA (https://www.rcsb.org/structure/unreleased/9PVA). ThT aggregation data, ddG calculations and cell-based aggregation data are available as Source Data. The raw data associated with these data are available on Zenodo under accession number 18665770. Any other data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. PBD id’s used in this study are: 6hre [http://doi.org/10.2210/pdb6HRE/pdb], 6nwp [http://doi.org/10.2210/pdb6NWP/pdb], 6tjo [http://doi.org/10.2210/pdb6TJO/pdb] and 7p65 [http://doi.org/10.2210/pdb7P65/pdb]. Code Availability The software used to analyze and determine a cryo-EM structure of the S320F295-330 fibril included CTFFIND v4.1, RELION v4.0 [https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page], COOT v9.4 [https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/]. All DDG calculations on the cryo-EM fibril structure were performed using an adapted flex_ddg protocol available on [https://git.biohpc.swmed.edu/s184069/flex_ddg_ala_scn_runner] using Rosetta v3.12 [https://www.rosettacommons.org/]. Flow cytometry data was analyzed using FlowJo v10 [https://www.flowjo.com/solutions/flowjo].