Chemical Interrogation and Reprogramming of ATAT1-Mediated Tubulin Acetylation

Acetylation of α -tubulin K40 by α -tubulin acetyltransferase 1 (ATAT1) using acetyl-coenzyme A (Ac-CoA) marks stable microtubule populations, yet chemical tools to directly measure ATAT1 ligand engagement, inhibit its activity, or visualize ATAT1-mediated modification on intact microtubules remain limited. Through the development of a quantitative binding assay, we uncovered that ATAT1 can bind unnatural cofactors but fails to efficiently use them in acyl-transfer reactions. Structure-guided mutation subsequently yielded ATAT1-L163A, which successfully installed clickable handles at the native α -tubulin K40 site of synthetic tubulin peptides, α -tubulin, and intact microtubules. Cu(I)-catalyzed azide–alkyne cycloaddition enabled visualization of modified microtubules by in-gel fluorescence and microscopy. Moreover, we report a p11-CoA bisubstrate inhibitor that suppressed both native acetylation and engineered acylation. Together, these tools provide chemically controlled access to ATAT1 activity and a site-verified, clickable K40 modification on intact microtubules.