Molecular Basis for Age-Dependent Acetylation by Tubulin Acetyltransferase

Microtubules are subject to a diverse array of posttranslational modifications. The majority of these modifications occur on the exterior surface of the microtubule. Acetylation of Lysine 40 is unique in that it occurs in the lumen of the microtubule. This modification is a marker for old, stable microtubules that are resistant to depolymerization. Tubulin acetyltransferase (TAT) is the enzyme responsible for this modification. Here we employ X-ray crystallography, electron microscopy, structure-based functional assays, single molecule imaging and first-principles modeling to understand how this unique enzyme is able to gain access to the microtubule and selectively modify microtubules that are long lived. Single molecule TIRF imaging of TAT-GFP reveals that TAT is undergoing 1-D diffusion along the microtubule and that the acetylation pattern is not biased for microtubule ends. The interaction time with the microtubule is unchanged when the microtubule surface is decorated with microtubule binding proteins or when the tubulin C-terminal tails are removed. However, TAT scanning is not observed when the luminal acetylation loop is missing. First-principles modeling demonstrate that the slow catalytic rate of the enzyme and not access to the luminal modification site is rate-limiting. Consistent with this, our X-ray structure of TAT reveals an active site not optimized for efficient catalysis. Thus, the rapid diffusion of TAT coupled with its low catalytic rate gives cells a mechanism for selectively marking long lived microtubules and evenly deposit the acetylation mark along their length. Our insights into TAT mechanism have broader implications for proteins that reside in the microtubule lumen.