Modifications of Lysine Residues 294 and 311 Within the Microtubule-Binding Region Alter Tau Aggregation and Tubulin Binding Equilibria.

Dysfunction of the microtubule-associated protein Tau is a central feature of Alzheimer's disease and related tauopathies, yet how site-specific lysine modifications modulate the functional and pathological states of Tau remains poorly understood. Here, we combine protein semi-synthesis with segmental isotope labelling and NMR spectroscopy of full-length Tau, to dissect how lysine acetylation and carboxymethylation within the microtubule-binding region of Tau regulate its interactions with tubulin, microtubules, and amyloid assembly. Site-specific modification at lysine 294 delays Tau-mediated tubulin polymerization and fibril formation, whereas acetylation at lysine 311 exerts more moderate effects, but alters fibril morphology. NMR spectroscopy of segmentally isotope-labelled Tau variants indicates that single lysine acetylation does not measurably weaken Tau binding to pre-formed microtubules; however, bivalent acetylation reduces microtubule binding, possibly by cumulative charge neutralization. Together, these results indicate that lysine acetylation redistributes Tau between functional states in a site- and valency-dependent manner. Our study helps to establish a mechanistic framework linking combinatorial lysine modifications to Tau dysfunction and highlights the utility of region-resolved structural approaches to decipher posttranslational modification-dependent equilibria in intrinsically disordered proteins.