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.